WO2025213014A1 - Compositions and methods for stabilizing mrna - Google Patents

Compositions and methods for stabilizing mrna

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Publication number
WO2025213014A1
WO2025213014A1 PCT/US2025/023146 US2025023146W WO2025213014A1 WO 2025213014 A1 WO2025213014 A1 WO 2025213014A1 US 2025023146 W US2025023146 W US 2025023146W WO 2025213014 A1 WO2025213014 A1 WO 2025213014A1
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mrna
methyltransferase
modified
rna
polypeptide
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French (fr)
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Kaifu CHEN
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Boston Childrens Hospital
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Boston Childrens Hospital
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01057—Methyltransferases (2.1.1) mRNA (nucleoside-2'-O-)-methyltransferase (2.1.1.57)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01166—23S rRNA (uridine2552-2'-O-)-methyltransferase (2.1.1.166)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01167—27S pre-rRNA (guanosine2922-2'-O-)-methyltransferase (2.1.1.167)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)

Definitions

  • the method involves contacting an mRNA with a heterologous 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA.
  • the present disclosure provides an in vitro method of stabilizing an 30 mRNA. The method involves contacting an mRNA with a 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA.
  • the present disclosure provides a method of stabilizing an mRNA.
  • the method involves contacting an mRNA with a 2’-O-methyltransferase polypeptide, such that an 35 internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA, where the 2’-O-methyltransferase polypeptide includes one or more of: cap 1 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • thermostabilizing mutations include one or more of: 20 R425X, R425C, G542X, G542V, Y639X, Y639G, Y639F, Y639V, H784X, H784A, H784G, H784S, E593G, V685A, I119V, G225S, K333N, D366N, F400L, S661G, F880Y, H772R, S430P, N433T, S633P, F849I, or P266L, where X is any amino acid.
  • the modified mRNA is characterized as having an increased half life of at least about 70% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 90% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at 10 least about 100% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 150% as compared to a reference mRNA.
  • the modified mRNA is characterized as having an increased half life of at least about 200% as compared to a reference mRNA. In any of the above aspects, or 15 embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 250% as compared to a reference mRNA.
  • the polynucleotide is an RNA molecule or a DNA/RNA hybrid. In any of the above aspects, or embodiments thereof, at least about 10% of the 20 ribonucleotides include a 2’-O-methyl modification.
  • the 2’-O-methyltransferase is meant an enzyme capable of modifying an RNA to comprise a 2’-O-methyl modification.
  • the RNA is an mRNA.
  • the 2’-O-methyltransferase is one or more of cap methyltransferase 1 (CMTR1), cap methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O- methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA 10 methyltransferase 3 (MRM3), tRNA guanosine 2
  • CMTR1 cap methyl
  • the alteration is an increase in the stability of a modified polynucleotide (e.g., 2x, 3x, 4x, 5x, 10x, 100x) relative to the stability of an unmodified polynucleoide (e.g., RNA)
  • a modified polynucleotide e.g., 2x, 3x, 4x, 5x, 10x, 100x
  • an unmodified polynucleoide e.g., RNA
  • analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • a polypeptide analog retains the biological activity of a 25 corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide.
  • biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding.
  • CMTR1 polypeptide a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_055865.1, or a fragment thereof, and having methyltransferase activity.
  • sequence of an exemplary CMTR1 polypeptide is provided below: 5 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • CMTR1 complementary metal-oxide-semiconductor
  • CMTR1 Homo sapiens cap methyltransferase 1
  • mRNA 20 GCAGTACTGGACCCGAGCGCGACGGTGCGGCTGGCGGACCCGGGCTGGCTTGTGGGGAAACGAAACTGAG
  • CMTR2 Homo sapiens cap methyltransferase 2
  • transcript variant 2 mRNA GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGGTGGGACTGGCTTCCCGGT GCCGCGAGGGCGGGTCCGGACAGCCTTCCCCCCAGTCCGGCACCATCTCCCTGCCTTGTGGCTGGAGG CGCCGCGGACCCAAAGGGAGGGACCATCCCGGGAAGCAGCCCCGAGAGCGGAAGTGCAGAATGGCTTCCT 15 CGAGAGAGTAAAGTGCAGCCTCTCCAGACACTGGGGCCCCAGTGGGCGTGGGCGAAGGTAATCCAGGCCT GGGTACGATTCCGGGCCCTCCTTCGACTTCCCAGCGGTTGCTGGTAGGAGGAGTTGGCGGAAGCACTTGG AACTCCTTTATAAGTGTCAGCTGTGAGATTTTAATTTGATTTGAAAAAA
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_018348.6
  • Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 1, mRNA GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGGTGGGACTGGCTTCCCGGT GCCGCGAGGGCGGGTCCGGACAGCCTTCCCCCCAGTCCGGCACCATCTCCCTGCCTTGTGGCTGGAGG 5 CGCCGCGGACCCAAAGGGAGGGACCATCCCGGGAAGCAGCCCCGAGAGCGGAAGTGCAGAATGGCTTCCT CGAGAGAGTAAAGTGCAGCCTCTCCAGACACTGGGGCCCCAGTGGGCGTGGGCGAAGTTGCTGGTAGGAG GAGTTGGCGGAAGCACTTGGAACTCCTTTATAAGTGTCAGCTGTGAGATTTTAATTTGATTTGAAAATGA GTAAGTGCAGAAAGACACCAGTTCAGCAGCTAGCAAGTCCCGCGTCAT
  • FBL Homo sapiens fibrillarin
  • FBLL1 Homo sapiens fibrillarin like 1
  • FBLL1 Homo sapiens fibrillarin like 1
  • tRNA cytidine(32)/guanosine(34)-2'-O)-methyltransferase isoform c [Homo sapiens] 5 MALNIATHVLKPGGCFVAKIFRGRDVTLLYSQLQVFFSSVLCAKPRSSRNSSIEAFAVCQGYDPPEGFIP DLSKPLLDHSYDPDFNQLDGPTRIIVPFVTCGDLSSYDSDRSYPLDLEGGSEYKYTPPTQPPISPPYQEA CTLKRKGQLAKEIRPQDCPISRVDTFPQPLAAPQCHTLLAPEMEDNEMSCSP >NP_036412.1 tRNA (cytidine(32)/guanosine(34)-2'-O)-methyltransferase isoform 10 a [Homo sapiens] MGRTSKDKRDVYYRLAKENGWRARSAFKLLQLDKE
  • FTSJ3 The sequence of an exemplary FTSJ3 polypeptide is provided below: >NP_060117.3 pre-rRNA 2'-O-ribose RNA methyltransferase FTSJ3 [Homo sapiens] MGKKGKVGKSRRDKFYHLAKETGYRSRSAFKLIQLNRRFQFLQKARALLDLCAAPGGWLQVAAKFMPVSS 5 LIVGVDLVPIKPLPNVVTLQQDITTERCRQALRKELKTWKVDVVLNDGAPNVGASWVHDAYSQAHLTLMA LRLACDFLARGGSFITKVFRSRDYQPLLWIFQQLFRRVQATKPQASRHESAEIFVVCQGFLAPDKVDSKF FDPKFAFKEVEVQAKTVTELVTKKKPKAEGYAEGDLTLYHRTSVTDFLRAANPVDFLSKASEIMVDDEEL AQHPATTEDIRVCCQDIRVLGRKELRSLLNWRTKLRRY
  • FTSJ3 The sequence of an exemplary FTSJ3 polynucleotide is provided below: 20 >NM_017647.4 Homo sapiens FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), mRNA AGATCCGGTCGCAGGCAGCGTCCAGCATGGACTGGTCCAGGCACCTCCGACCCACCCGACGCTCCTGCCC CGGCCCGCTCTCCACTCAGATCACGCTCGCTCCGCACCCAGCCCCGTAACCTGCCGGGCCGCCACTGCAC CCGCCATACCTGCTCTGGTCCGTCAAGCGCCATCTTCTCTTCAGCAGAGACCGCCGGCATCCGAGCCG TCTTGGCGCGCAAGCGCGGAAGCGGAGGATTGGGACCTGAAGACCAGGCAGAGAAGAATTCCGGGTCAAT 25 GGGCGGGGTGTGCGGCGAGACCACACTAAGGGAACCATGATGGGAGGGGCGGCTACTCGAGTTCAGGGGC TTTGGAAGGATGAGGAAGGAGTAGAACTCCTCCT
  • RNA_001096062.1 or NP_653185.2 or a fragment thereof, and having methyltransferase activity. 35
  • the sequences of exemplary HENMT1 polypeptides are provided below: >NP_001096062.1 small RNA 2'-O-methyltransferase [Homo sapiens] MEENNLQCSSVVDGNFEEVPRETAIQFKPPLYRQRYQFVKNLVDQHEPKKVADLGCGDTSLLRLLKVNPC IELLVGVDINEDKLRWRGDSLAPFLGDFLKPRDLNLTITLYHGSVVERDSRLLGFDLITCIELIEHLDSG DLARFPEVVFGYLSPSMIVISTPNSEFNPLFPSVTLRDSDHKFEWTRMEFQTWALYVANRYDYSVEFTGV 40 GEPPAGAENVGYCTQIGIFRKNGGKATESCLSEQHDQHVYKAVFTTSYPSLQQERFFKLVLVNEVSQQVE 28 ATTORNEY
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 SLRVSHLPRRKEQAGERGDKPKDIGGSKAPVPCFGPVFTEVEKAKIENSPTPFCVGDKFFVPLQRLLAYP KLNRLCANEEMMRSVIADSIPLSSDGSAVVADLRNYFDEQFEF >NP_653185.2 small RNA 2'-O-methyltransferase [Homo sapiens] 5 MEENNLQCSSVVDGNFEEVPRETAIQFKPPLYRQRYQFVKNLVDQHEPKKVADLGCGDTSLLRLLKVNPC IELLVGVDINEDKLRWRGDSLAPFLGDFLKPRDLNLTITLYHGSVVERDSRLLGFDLITCIELIEHLDSG DLARFPEVVFGYLSPSMIVISTPNSEFNPLFPSVTLRDSDHKFEWTRMEFQTWALYVANRYDYSVEFTGV GEPPAGAENVGYCTQIGIFRKNGGKATESCLSEQHD
  • HENMT1 Homo sapiens HEN methyltransferase 1
  • transcript variant 1 mRNA AAGCCGCCTTCCGGCTCGCGGTCTGCGGACGCGCTCGGAGTTGGGGGCCTTCGCCAACAGCTGCCGTACC 5 GTGCCGCGTCGCAGCCGCACGCTAGCGGGGTCAACGGCGCGGCAGGCGCAGGGGTCCTCGCCAAGGTTCG GAAGAGGACCTTGAAAATGGCTTCTGGGCATGCGCCACAGCGGCCGTTACGAGACGCAAAACCGACGCCT TGGAACTGAGAGCCTAGAGGGACGGAGCTCGGCGCACCATGAGGCTCCCGGGGCCCAAGGCCGACCGTGA GAGCGCACGAGCTGAGTCAGTGCTGATTCTCTCAAATAGAGCTTGAAGGATAAATCTTCATTTTTGTTTC AACAAAACTTCGAAACAAAATGGAAGAAAATAATCTACAGTGCAGTAGTGTGTGT
  • the source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species.
  • a heterologous or exogenous nucleic acid molecule i.e., not endogenous or native
  • chromosomal genetic material e.g., as a plasmid or other form of self-replicating vector
  • chromosomal genetic material e.g., as a plasmid or other form of self-replicating vector
  • heterologous may also refer to a non-native enzyme, protein, polypeptide, or other activity, (e.g., directly introduced or encoded by an exogenous nucleic acid molecule) introduced into the host cell, even if the host cell encodes a homologous 5 protein or activity.
  • a cell comprising a "modification” or a “heterologous" polynucleotide or binding protein includes progeny of that cell, regardless of whether the progeny were themselves transduced, transfected, or otherwise manipulated or changed.
  • a “host cell” or “cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that 10 have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • Hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • adenine and thymine are complementary nucleobases that pair through the formation of 15 hydrogen bonds.
  • isolated denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate 31 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • isolated polypeptide is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In an embodiment, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the disclosure.
  • MRM1 polypeptide The sequence of an exemplary MRM1 polypeptide is provided below: >NP_079140.2 rRNA methyltransferase 1, mitochondrial precursor [Homo sapiens] MALLSTVRGATWGRLVTRHFSHAARHGERPGGEELSRLLLDDLVPTSRLELLFGMTPCLLALQAARRSVA 20 RLLLQAGKAGLQGKRAELLRMAEARDIPVLRPRRQKLDTMCRYQVHQGVCMEVSPLRPRPWREAGEASPG DDPQQLWLVLDGIQDPRNFGAVLRSAHFLGVDKVITSRRNSCPLTPVVVSKSSAGAMEVMDVFSTDDLTGF LQTKAQQGWLVAGTVGCPSTEDPQSSEIPIMSCLEFLWERPTLLVLGNEGSGLSQEVQASCQLLLTILPR RQLPPGLESLNVSVAAGILLHSICSQRKGFPTEGERRQLLQDPQEPSARSEGLSMAQHPGLSSGPEKERQ NEG 25
  • MRM1 Homo sapiens mitochondrial rRNA methyltransferase 1 (MRM1), 30 mRNA; nuclear gene for mitochondrial product GAACCCGGAAGCGAGGGACCCACGTGGGAGCCTGGGAGCGGGTGGTCGTAGCTCGGTAGTCCAGTTGTGG GTAATCGGGGCTGTTTGTTCCTGTCCGAGACGGCTGTCGAGTACCCTTCACCTCGG TGTTGGGAGCCTGGGAGCGAACTGCGGCGCGGGTTACCGCTCCCGGGGACGCAGCAAGGGGCATCGAGTC CCTGGCGGGAGCTGCGCCATGGCATTGCTCTCGACCGTCCGGGGCGCGACCTGGGGTCGCCTCGTCACCC 35 GTCATTTCTCCCATGCAGCGCGGCATGGGGAGCGGCCTGGTGGGGAGGAGCTAAGCCGCTTGCTGCTGGA TGACCTGGTGCCGACCTCTCGGCTGGAGCTTCTGTTTGGCATG
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGCTCCGGATGGCCGAGGCGCGGGACATTCCAGTTCTGCGGCCCAGACGGCAGAAACTGGACACAATGTG CCGCTACCAGGTCCACCAGGGTGTCTGCATGGAGGTGAGCCCGCTGCGGCCCCGGCCTTGGAGAGGCC GGGGAGGCGAGCCCAGGCGACGACCCCCAGCAGTTGTGGCTCGTCCTCGATGGGATCCAGGATCCCCGGA ATTTTGGGGCTGTGCTGCGTTCCGCACACTTCCTCGGAGTGGATAAGGTCATCACCAGCCGGAGAAACAG 5 CTGCCCGCTCACTCCAGTAGTCAGCAAGTCCAGCGCGGGGGCTATGGAGGTGATGGACGTGTTCTCCACT GATGACCTCACCGGATTTTTACAGACCAAAGCCCAGCAGGGCTGGCTCGTGGCCGGCACGGTGGGCTGCC CAAGCACAGAGGATCCAGTCCTCCCATCATCATGAGTTGCTTGCTTGCTTGCTCC
  • NP_001304876.1 or NP_060616.1, or a fragment thereof, and having methyltransferase activity The sequences of exemplary MRM3 polypeptides are provided 30 below: >NP_001304876.1 rRNA methyltransferase 3, mitochondrial isoform 2 [Homo sapiens] MTYPKTQLQHSLPLLLICDNLRDPGNLGTILRSAAGAGCSKVLLTKGCVDAWEPKVLRAGMGAHFRMPII NNLEWETVPNYLPPDTRVYVADNCGLYAQAEMSNKASDHGWVCDQRVMKFHKYEEEEDVETGASQDWLPH 35 VEVQSYDSDWTEAPAAVVIGGETYGVSLESLQLAESTGGKRLLIPVVPGVDSLNSAMAASILLFEGKRQL RGRAEDLSRDRSYH >NP_060616.1 rRNA methyltransferase 3, mitochondrial isoform 1 precursor [Homo sapiens] 40 MAALVRPARFVVR
  • MRM3 mitochondrial rRNA methyltransferase 3
  • MRM3 mitochondrial rRNA methyltransferase 3
  • mRNA nuclear gene for mitochondrial product GTGACGCAGCCCGGGTCTCAGGGAACATGGCGGCGCTGGTGAGACCCGCGAGGTTTGTCGTGCGACCGTT GCTGCAGGTGGTCCAGGCTTGGGACCTTGACGCGAGGCGCTGGGTCCGGGCGCTGCGGCGGAGCCCAGTG AAAGTGGTGTTTCCTTCCGGAGAGGTGGTGGAACAGAAGCGCGCTCCTGGGAAGCAGCCCCGCAAGGCAC 15 CATCTGAGGCCAGTGCCCAGGAGCAACGAGAGAAACAACCGCTCGAGGAGTCCGCATCCCGCGCTCCCAG CACCTGGGAAGAGTCTGGGCTTCGCTACGATAAAGCTTATCCCGGGGACAGGAGGCTGAGGGATTTTTGC CAAGCCTGACCATGTTAAGATGACATATCCAA
  • the modified polynucleotide is an RNA (e.g., mRNA) or a DNA/RNA hybrid.
  • the mRNA is chemically synthesized or in vitro transcribed.
  • the modified mRNA comprises one or more internal nucleobases comprising a 2’-O-methyl modification.
  • the 30 modified mRNA comprises a modification at a position between the 5’ and 3’ termini (i.e., a modification at an internal position) and shows increased stability relative to a reference polynucleotide.
  • operably linked refers to a functional linkage between a regulatory sequence and a coding sequence, where a first polynucleotide is positioned adjacent to a second polynucleotide 35 that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
  • appropriate molecules e.g., transcriptional activator proteins
  • the described components are therefore in a relationship permitting them to function in their intended manner. For example, placing a coding sequence under regulatory control of a promoter means 36 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • portion is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the 5 reference nucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.
  • positioned for expression is meant that the polynucleotide of the disclosure (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a polynucleotide or 10 polypeptide described herein).
  • promoter refers to a sequence of DNA that directs the expression (transcription) of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene.
  • a promoter may be “inducible”, initiating transcription in response to an inducing agent or, in contrast, a promoter may be “constitutive”, whereby an inducing agent does15 not regulate the rate of transcription.
  • a promoter may be regulated in a tissue-specific or tissue- preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types.
  • “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. 20 By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • reference is meant a standard or control condition.
  • a reference polynucleotide is an unmodified or wild type version of a polynucleotide (e.g., mRNA) having the same or substantially the same sequence as a modified mRNA.
  • the reference polynucleotide e.g., mRNA
  • a "reference sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an 5 endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof.
  • nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having “substantial 10 identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, and less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency 20 hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, or at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels 25 of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM 30 trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing 38 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 temperature.
  • stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C.
  • 5 wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Hybridization techniques are well known to those skilled in the art and 10 are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al.
  • such a sequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for 20 comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs).
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs.
  • Such software matches identical or similar 25 sequences by assigning degrees of homology
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a BLAST program may be used, with a 30 probability score between e -3 and e -100 indicating a closely related sequence.
  • upS pre-rRNA (guanosine2922-2'-O)-methyltransferase polypeptide or “SPB1 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_009877.1, or a fragment thereof, and having methyltransferase activity.
  • the sequence of an exemplary SPB1 polypeptide is provided below: 39 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_009877.127S pre-rRNA (guanosine2922-2'-O)-methyltransferase [Saccharomyces cerevisiae S288C] MGKTQKKNSKGRLDRYYYLAKEKGYRARSSFKIIQINEKYGHFLEKSKVVIDLCAAPGSWCQVASKLCPV NSLIIGVDIVPMKPMPNVITFQSDITTEDCRSKLRGYMKTWKADTVLHDGAPNVGLGWVQDAFTQSQLTL 5 QALKLAVENLVVNGTFVTKIFRSKDYNKLIWVFQQLFEKVEATKPPASRNVSAEIFVVCKGFKAPKRLDP RLLDPKEVFEELPDGQQNMESKIYNPEKKVRKRQGYEEGDNLLYHETSILDFVRTEDPISMLGEMNKFTI DENDHEWKILKKLKQTTDEFRSCIEDLK
  • SPB1 polynucleotide The sequence of an exemplary SPB1 polynucleotide is provided below: >NM_001178698.1 Saccharomyces cerevisiae S288C 27S pre-rRNA (guanosine2922- 20 2'-O)-methyltransferase (SPB1), partial mRNA ATGGGTAAGACACAAAAGAAGAATAGTAAGGGACGTTTAGATAGATACTATTATCTAGCAAAGGAGAAAG GTTATCGTGCTCGTTCATCCTTTAAGATTATTCAGATTAATGAAAAGTATGGCCACTTCTTAGAAAAATC GAAGGTTGTTATTGATCTGTGCTGCTCCTGGTTCATGGTGTCAAGTTGCATCCAAACTCTGTCCTGTC AACTCCTTAATTATTGGTGTTGATATTGTTCCAATGAAGCCGATGCCCAATGTTATAACTTTTCAAAGTG 25 ATATTACAACAGAAGATTGTAGATCAAAATTGAGGGGTTATATGAAGACTTGGAAAGCTGATACAGTGTT
  • Ranges provided herein are understood to be shorthand for all of the values within the 20 range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • tRNA guanosine 2 -O-methyltransferase polypeptide or “TARBP1 polypeptide” is 25 meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No.
  • TARBP1 [Homo sapiens] MEWVLAEALLSQSRDPRALLGALCQGEASAERVETLRFLLQRLEDEEARGSGGAGALPEAAREVAAGYLV 30 PLLRSLRGRPAGGPDPSLQPRHRRRVLRAAGAALRSCVRLAGRPQLAAALAEEALRDLLAGWRAPGAEAA VEVLAAVGPCLRPREDGPLLERVAGTAVALALGGGGDGDEAGPAEDAAALVAGRLLPVLVQCGGAALRAV WGGLAAPGASLGSGRVEEKLLVLSALAEKLLPEPGGDRARGAREAGPDARRCWRFWRTVQAGLGQADALT RKRARYLLQRAVEVSAELGADCTCGPQEGNGPSLFWWSERKKDELLKFWENYILIMETLEGNQIH
  • TARBP1 The sequence of an exemplary TARBP1 polynucleotide is provided below: >NM_005646.4 Homo sapiens tRNA guanosine 2 -O-methyltransferase TARBP1 (TARBP1), mRNA 20 CGGTCCTGTGCGCACGCATCGCACACGCCGGCGCCTTCCTTTGGGAGCCCGGGCCGGTGGCGCGGGCGCT CGGCAAATGGAGTGGGTGCTCGCGGAAGCGCTGCTCTCGCAGAGCCGGGACCCCCGGGCCCTGCTTGGGG CGCTGTGCCAAGGGGAGGCATCCGCGGAGCGCGTGGAGACGCTGCGCTTCCTTCTGCAGCGGCTCGAGGA CGAGGAGGCGCGCGGCAGCGGGGGCGCAGGCGCGCTCCCGGAGGCGGCGCGCGAGGTGGCTGCAGGGTAC CTCGTGCCACTGCTGCGGAGCCTGCGGACGCCCCGCGGGCGGCCCGGACCCCAGTCTGCAGCCTCGCC 25 ACCGCCGGCGTGC
  • tRNA methyltransferase 11 homolog polypeptide or “TRMT11 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos.
  • TRMT11 The 40 sequences of exemplary TRMT11 polynucleotides are provided below: 47 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_001031712.3 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 1, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT 5 TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCT TAAAAACTACCCTGTGGAAG
  • TRMT13 The sequences of exemplary TRMT13 polynucleotides are provided below: 65 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_001393409.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), transcript variant 2, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC 5 TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC AAGATATTAATGCAGGCTTA
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ATGAGATAACACAAGTAAAATGCATAGAACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAGTA ATAGTAGTGGTAGTAACTCGTGAATCTTTTTAATAACATAATAGGCTTTGATTTTATTATCTCTTTAAGT TGTTAACTTTTTTCCCTTGTTATAAGTTTTATGTCAAGTAAGGTAGTTTGTTTAAGTTAGTTACCCATGT CCCCAATCAAGGGAACCTAAATGAAGATATATCATTAAATAATTAACCTTTTTTTATTGTGTCAAGCAAT 5 ACTAATTTGTGTCATAAAATTTGTTTTGCATCATTCAACAATTTATGCCCAGAAATAAACAATATTCTAC AACAGG >NM_001393410.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), transcript variant 3, mRNA 10 AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCCTGGTTTTCCAGACC
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ATGCACAAGAAAATACTAAACCAAATATATGGTAGGACTGTTATTCTCTTCTGCATCTTATCCCTATTTT GTTTCTGCCTTTATTTGTAAAAATTGTCTTAATTGATGGTACATTTGCCAAGACAAAGGTTCAGAATTAC TAATTTTAGATATTATGATATTCTGAAATAACTATTTTTATCCTGTAGTTCTATGATTATATGATTTGTA AATAAGAAGCCTAACCAATTTAAAATTCCTGACTTTAGTCTCATATATCTTGTCAGACTGTCTCGGTTCA 5 AATTCCAAACCTACCATCTTCAGTTGTGCGACCTTGGGCATGCTACCTAATCTCTTTGTGCCTCAATTTC CTCCTTAAAATGGGGATGATGATGATAACAATAATACCTACCTCACAAGGTTGTTGTGAGCATCAAAT GAGATAACACAAGTAAAATGCATAGAACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAG
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TAACCTTTTTTTATTGTGTCAAGCAATACTAATTTGTGTCATAAAATTTGTTTTGCATCATTCAACAATT TATGCCCAGAAATAAACAATATTCTACAACAGG >NM_001393412.1
  • Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), 5 transcript variant 5, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC 10 AAGATATTAATGCAGG
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GTGAAGAGCATGATGATGGAGGATACAGAATCACAGATGATGGCGCTGATTGTTTGCCTGGGTAAGAGAC TACTTTTGTAATGCATGATACTAAAGGAGAAATATTATATTGTACTGTACTTTAGATGACTATTATCAAC AATTCATGAAAATGTATTTTATAATCTAATTTTAGAATAAGCTAAAATATACACATCTTTTATTGTGGTC ATAAATATAATGTGTCTTGGAAGCATGGGACAAAGTACTTAGTACATTATGGGTTAGTTAACAGTCTCTC 5 AATAAATAGTGACAAAAATAATTTTTTATAAACTTTTGCAAAATTTTAACAATGTTTTGTGTGTGTTTAT TCAATTTAGGCTTCTTAGTGTTGAAGAAAAGAAGAAAATAGGGCATCTTTGTAAATTGCTGATTGACCAA GGTCGAATCCAGTATTTGCAGCAGAAGGGATTCAGTCCTGCTTTGCAGTACTATAC
  • tRNA methyltransferase 44 homolog polypeptide or “TRMT44 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001337162.1 or NP_689757.2, or a fragment thereof, and having methyltransferase activity.
  • TRMT44 polypeptides are provided 5 below: >NP_001337162.1 probable tRNA (uracil-O(2)-)-methyltransferase isoform 3 [Homo sapiens] MCIKFSSVIAKKNERWHSDGIVYPKPTWLGEELLAKLAKWSVENKKSDFKSTLSLISIMKYSKAYQELKE KYKEMVKVWPEVTDPEKFVYEDVAIAAYLLILWEEERAERRLTARQSFVDLGCGNGLLVHILSSEGHPGR 10 GIDVRRRKIWDMYGPQTQLEEDAITPNDKTLFPDVDWLIGNHSDELTPWIPVIAARSSYNCRFFVLPCCF FDFIGRYSRRQSKKTQYREYLDFIKEVGFTCGFHVDEDCLRIPSTKRVCLVGKSRTYPSSREASVDEKRT QYIKSRRGCPVSPPGWELSPSPRWVAAGSAGHCDGQQALDARVGCVTRAWAAEHGAGPQA
  • TRMT44 Homo sapiens tRNA methyltransferase 44 homolog
  • 35 transcript variant 3 mRNA GTCATCTCGGCGCGCCGCTGCCAGGGCTGTACACCTGCTGGCTGCCATGGCTGAGGTGGGCCGTACCGGG ATCAGCTACCCAGGCGCGCTTCTCCCACAGGGCTTCTGGGCTGCGGTCGAAGTGTGGCTGGAGAGGCCGC AGGTGGCAAACAAACGGCTTTGCGGCGCCCGCCTGGAGGCCCGCTGGAGCGCCGCCCTGCCCTGCGCGGA GGCCCGCGCGCCCGGGACTAGCGCAGGCTCGGAGCAGAAGGAGCGGTCCGGGACCCGGCCAGGGTTCC 75 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF.
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CCCGGAGGGGGCCCGGGTCCCAGGTCGCTATCAGGACCCGAGCAGGGCACGGCATGTTGCGAACTTGAGG AGGCCCAGGGCCAGTGCCAGCAAGAGGAGGCACAGAGGGAAGCCGCCTCAGTGCCCCTGAGGGACTCCGG GCACCCCGGCCATGCTGAAGGAAGGGAGGGCGACTTCCCCGCCGCAGATCTGGATTCGCTTTGGGAGGAT TTCTCCCAAAGTCTCGCCCGTGGCAATTCGGAGTTGCTGGCCTTCCTCACCAGCTCCGGGGCGGGATCGC 5 AGCCAGAGGCGCAGCGTGAGCTCGACGTGGTTCTCAGAACCGTCATCCCGAAAACTAGCCCACATTGCCC CCTTACAACTCCCAGGAGGGAAATAGTCGTGCAAGATGTCCTCAATGGAACCATAACGTTTTTGCCTTTG GAAGAAGATGATGAGGGGAACCTAAAGGTTAAGATGAGCAATGTGTG
  • a vector is meant a nucleic acid molecule, for example, a plasmid, cosmid, virus, or bacteriophage that is capable of replication in a host cell.
  • a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, 5 bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell.
  • expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.
  • the term “about” is 10 understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • the recitation of a listing of chemical groups in any definition of a variable herein 15 includes definitions of that variable as any single group or combination of listed groups.
  • the recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
  • FIGs.1A-1H provide a drawing, graphs, plots, a picture, and a heat map showing that 2’- O-methylation (Nm) at internal sites on mRNA and the expression of Nm writer protein FBL is 25 required for the greater stability of 2′-O-methylated mRNAs compared to the rest of mRNAs in HEK293T cells.
  • FIG.1A shows Nm (top panel) and related questions on RNA stability (bottom).
  • FIG.1B is a gene set enrichment analysis (GSEA) showing a relationship between 2′-O- Methylation and mRNA half-life.
  • GSEA gene set enrichment analysis
  • FIG.1C is a cumulative graph and boxplot to show the half- life of mRNAs with or without detectable Nm. Boxplot to show the half-life of mRNAs with or 30 without Nm. The top line in the graph is Nm(-) and the bottom line is Nm(+).
  • FIG.1D is a Western blot to show the knockdown efficiency of FBL and NOP56 in C4-2 cells.
  • FIGs.1E-1F show the cumulative fraction of mRNAs plotted against the change of mRNA half-life in response to knockdown of FBL (FIG.1E) and NOP56 (FIG.1F).
  • the top line is Nm(+) and the bottom line is Nm(-).
  • FIG.1G is a boxplot to show the half-life of 2′- 35 O-methylated mRNAs under individual conditions.
  • FIG.1H is a heatmap to show half-life of top 78 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • FIGs.2A-2J provide graphs, plots, a schematic, and drawings showing that the binding of FBL is required for the greater stability of FBL-binding mRNAs when compared to the rest of the mRNAs in HEK293T cells.
  • FIG.2A is a bar plot showing the overlap of 2′-O-methylated mRNAs and FBL-binding mRNAs. The top portion of each bar shows percentage of no binding, 10 and the bottom portion shows percentage binding.
  • FIG.2B shows the cumulative fraction of mRNAs plotted against half-life of mRNAs with or without detectable binding of FBL. The top line is FBL-binding(-) and the bottom line is FBL-binding(+).
  • FIG.2C shows the cumulative fraction of mRNAs plotted against half-life of mRNAs with high or low binding intensity of FBL.
  • the 15 top line is FBL-binding low and the bottom line is FBL-binding high.
  • the inset box plot shows FBL-binding low on the right, and FBL-binding high on the left.
  • FIGs.2D-2E show the cumulative fraction of mRNAs plotted against the change of mRNA half-life in response to knockdown of FBL (FIG.2D) and NOP56 (FIG.2E).
  • FIG.2F is a boxplot to show the half-life of 20 FBL-binding mRNAs under three different conditions.
  • FIG.2G is an RTL-P assay which detected Nm levels on four mRNAs (ACTG1, SNHG5, CALR, PHGDH) under control, siFBL, and siNOP56 conditions.
  • the cartoon in the top left subpanel indicates the locations of primers used for RTL-P assay; m: Nm site; Um: upstream of Nm site; Dm: downstream of Nm site; F: forward primer; R: reverse primer; RT: Reverse transcription.
  • FIG.2I is the relative expression level of mRNA plotted against time after inhibition of transcription in each panel. 30 The lines in each graph are ordered from top to bottom, as follows: siCTRL, siNOP56, siFBL.
  • FIGs.3A-3J are plots, graphs, and a pie chart showing that elevated FBL expression correlated with expression upregulation of FBL-binding and 2’-O-methylated mRNAs in 5 prostate cancer (PCa)( PCa) cells.
  • FIG.3A is a boxplot showing RNA expression level of FBL, NOP56, and NOP58 in prostate normal and cancer samples from the TCGA project.
  • FIG.3B shows that the genome browser tracks showing RNA-Seq read density at individual gene loci of C4-2 and PrEC cells.
  • FIG.3C is a pie chart showing the distribution of FBL-binding sites in individual categories of RNAs in C4-2 cells.
  • FIG.3D is a GSEA result which shows the 10 relationship between the binding of FBL on mRNA and the expression change of mRNA in response to FBL knockdown.
  • FIG.3E is a boxplot to show expression change of mRNAs in response to FBL knockdown.
  • FIG.3F shows the cumulative fraction of mRNAs plotted against the change of mRNA half-lives in response to FBL knockdown in C4-2 cells. The top line is FBL-binding(+) and the bottom line is FBL-binding(-).
  • FIG.3G is a boxplot to show mRNA 15 half-life of FBL-binding mRNAs under two conditions in C4-2 cells.
  • FIG.3H shows the cumulative and boxplot of half-life change of mRNAs between C4-2 and PrEC cells. The top line is C4-2 FBL-binding(-) and the bottom line is C4-2 FBL-binding(+).
  • FIG.3I is a GSEA to show the relationship between the binding of FBL on mRNA in C4-2 cells and the expression change of mRNA between C4-2 and PrEC cells.
  • FIG.3J is a boxplot showing expression change 20 of mRNAs between C4-2 and PrEC cells.
  • FIGs.4A-4K are a schemtica, graphs, plots, and a pie chart showing a machine learning model for detection of Nm based on Nanopore direct RNA-seq.
  • FIG.4A is a cartoon showing 25 the flowchart of a machine learning model to detect Nm based on Nanopore direct RNA-seq.
  • FIG.4B are receiver operating characteristic (ROC) curves which demonstrate the performance of the machine learning model for detecting known Nm sites in the human thyroid carcinoma cell line of KTC-1. The two panels were plotted for two different 5-mers.
  • FIGs.4C-4D are bar plots to show the Nm modification ratio defined by the machine learning model at individual 30 sites on the 28S (FIG.4C) and 18S (FIG.4D) rRNAs of C4-2 cells.
  • FIGs.4E-4F are boxplots to show Nm modification ratio defined by the machine learning model at individual sites on 28S rRNA (FIG.4E) and 18S rRNA (FIG.4F) in C4-2 cells under siCTRL and siFBL conditions.
  • FIG.4G shows rRNA Nm modification ratio at known snoRNA-targeted Nm sites on 25S rRNA in yeast under wild type, snR60 knockout, snR61 knockout, and snR62 knockout conditions.
  • the bars in each set of four are ordered, from top to bottom, as follows: WT; snR60_KO; snR61_KO; snR62_KO.
  • FIGs.4H-4I is a boxplot to show Nm modification ratio defined by the machine learning model at individual sites on 28S rRNA (FIG.4H) and 18S rRNA (FIG.4I) in Drosophila cells under siCTRL and siFBL conditions.
  • FIG.4J is a probability density plot of 5 Nm modification ratio for mRNAs and rRNAs in C4-2 cells. The left peak is mRNA and the right peak is rRNA.
  • FIG.4K is a pie chart that shows the distribution of Nm sites in different sequence regions in C4-2 cells. P values were determined by two-tailed unpaired Wilcoxon’s tests (FIGs.4E, 4F, 4H, 4I).
  • FIGs.5A-5M are graphs, plots, and a diagram showing that Nm detected on mRNA by 10 machine learning model based on Nanopore direct RNA-seq is downregulated by FBL knockdown in C4-2 cells.
  • FIG.5A is a boxplot to show the Nm ratio of Nm sites on mRNAs.
  • FIG.5B is a scatter plot to show the Nm ratio of individual Nm sites on mRNAs in control and FBL knockdown cells.
  • FIG.5C is an average density plot of Nm sites on mRNAs. The top line is siCTRL and the bottom line is siFBL.
  • FIG.5D is a Venn diagram to show overlap between15 FBL-binding and 2′-O-methylated mRNAs.
  • FIG.5E is a bar plot that shows the percentage of 2’- O-methylated mRNAs for individual gene groups ranked by mRNA expression level from the lowest on the left side to the highest on the right side.
  • FIG.5F is a GSEA that shows the relationship between mRNA Nm modification and expression change upon knockdown of FBL.
  • FIG.5G is a boxplot showing expression change of individual mRNA groups upon FBL 20 knockdown.
  • FIG.5H is a boxplot showing mRNA half-life of individual mRNA groups.
  • FIG.5I shows the cumulative fraction of mRNAs plotted against degree of mRNA half-life change upon FBL knockdown. The lines are ordered, from top to bottom, as follows: Nm Hypo; Nm(+); Nm(-).
  • FIG.5J is a boxplot showing mRNA half-life change of individual mRNA groups in response to knockdown of FBL.
  • FIG.5K is a boxplot showing the half-life of 2′-O-methylated25 and unmethylated noncoding RNAs.
  • FIG.5L is a boxplot showing expression change of 2′-O- methylated noncoding RNAs in C4-2 cells.
  • FIG.5M is a boxplot showing mRNA half-life of noncoding RNAs 2′-O-methylated in C4-2 cells. P values were determined by two-tailed unpaired Wilcoxon’s tests (FIGs.5A, 5G, 5H, 5J-M) and K-S test (FIG.5I).
  • FIGs.6A-6P are heatmaps, graphs, plots, and pictures showing that FBL expression in PCa cell C4-2 is required for the Nm modification and expression of mRNAs in cancer pathways.
  • FIG.6A is a heatmap to show Nm ratio of 10 example Nm sites (on 8 mRNAs) that show down-regulation of Nm in Nanopore direct RNA-seq data upon FBL knockdown in C4-2 cells.
  • FIG.6B is an RTL-P assay to detect the Nm level at 7 Nm sites of mRNAs shown in (FIG. 81 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 6B Total RNAs from control, FBL-deficient, and NOP56-deficient C4-2 cells were extracted and subjected to reverse transcription (RT) with RT primer at low (1 ⁇ M) or high (1 mM) concentration of dNTP, respectively. The obtained cDNA was then amplified with primer pairs corresponding to upstream (Um) or downstream (Dm) regions of the methylation site.
  • FIG.6C is 5 a densitometric analysis of data from FIG.6B shown as signal intensity ratio of PCR products at low dNTP (1 ⁇ M) over high dNTP (1 mM) level. Methylation levels in control cells were set close to 1.
  • FIG.6D is a heat map to show the mRNA expression level of the 8 example genes.
  • FIG.6E is a boxplot showing 10 expression change of C4-2 cell 2′--O-methylated mRNAs in PCa and normal patients from the TCGA project dataset.
  • FIG.6F is a Western blot showing the protein levels of PSMD13 in each group as indicated.
  • FIGs.6G-6H are RTL-P assays to detect the Nm level at two Nm sites of PSMD13 in each group as indicated.
  • FIG.6I is a boxplot showing the expression of PSMD13 in the TCGA prostate cancer patients grouped based on nodal metastasis status.
  • FIG.6J shows 15 disease-free survival of patients with different PSMD13 expression levels in PCa samples from the TCGA dataset. The top line is Low and the bottom line is High.
  • FIG.6K is a Western blot to show the knockdown efficiency of PSMD13 in C4-2 cells.
  • FIG.6L is a CellTiter assay to measure the effect of PSMD13 knockdown on cell proliferation. The lines are ordered, from top to bottom, as follows: siCTRL; siPSMD13-1; siPSMD13-2.
  • FIGs.7A-7N are graphs, plots, a diagram, a picture, and a drawing showing that mRNA stabilization by Nm is associated with 3′ UTR shortening.
  • FIG.7A is a cumulative fraction plot of 3′ UTR lengths of individual mRNAs in three groups. The lines are ordered, from top to bottom, as follows: Nm Hypo; Nm(+); Nm(-).
  • FIG.7B is a box plot to show the number of miRNA target sites in 3′ UTR regions of individual mRNAs in three groups.
  • FIG.7C is a box 30 plot to show ARE scores for 3′ UTR regions of individual mRNAs in three groups.
  • FIG.7D is a scatter plot to show PDUI values of individual mRNAs in control and FBL knockdown conditions. The internal bar plot shows numbers of 3′ UTR lengthening and shortening events induced by FBL knockdown.
  • FIG.7E is a Venn diagram that shows the overlap between genes showing lengthened 3′UTR upon siFBL and 2′-O-methylated genes under control condition.
  • FIG.7F is a bar plot to show the number of overlaps observed or expected by chance between genes that show lengthened or shortened 3′ UTR upon siFBL and genes that show 2′-O- methylated mRNAs under control condition. The left bar in each set of two is Overlap, and the right bar is Expected.
  • FIG.7J is a bar plot that shows the percentage of Nm sites overlapping with the binding regions of 10 different RNA binding proteins (RBPs) in HEK293T 15 cells. The top bar in each set of two is Overlap, and the bottom bar is Expected.
  • FIG.7K is a scatter plot showing a correlation between the PDUI changes induced by FBL and CPSF depletion in HepG2 cells.
  • FIG.7L shows RIP-qPCR to detect the change of the binding of CPSF7 to 2′-O-methylated mRNAs upon FBL knockdown.
  • FIG.7M is a Western blot to 20 show the expression of individual proteins under individual conditions.
  • FIG.7N is a cartoon to show the working model of Nm-dependent regulation of RNA stability.
  • FIGs.8A-8I provide a flow chart, graphs, a diagram, plots, and a heatmap showing an 30 analysis of half-life, Nm, and binding of FBL in mRNAs of HEK293T cells.
  • FIG.8A is a flow chart of RNA-seq experiment to profile RNA half-life.
  • FIG.8B is a bar plot that shows the expression of FBL and NOP56 determined by RT-qPCR in the control and knockdown cells.
  • FIG.8C shows that the genome browser tracks showing RNA-seq read density at the FBL and NOP56 loci.
  • FIG.8D is a Venn diagram showing the overlap between FBL-binding and 2′-O- 83 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 methylated mRNAs.
  • FIG.8E is a cumulative plot and boxplot showing half-life of 2′-O- methylated and unmethylated mRNAs. The top line is Nm(+) and the bottom line is Nm(-).
  • FIG.8F is a boxplot showing half-life of mRNA with or without detectable FBL binding.
  • FIG. 8G is a boxplot showing half-life of mRNAs with high or low binding intensity of FBL.
  • FIG.8H 5 is a heatmap to show half-life of FBL-binding mRNAs; 50 mRNAs ranked on top based on the degree of half-life decrease upon FBL knockdown were shown.
  • FIG.8I shows Genome browser tracks showing RNA-seq read density at the loci of 2′-O-methylated mRNAs. P values were determined by two-tailed Student’s t-test (FIG.8B), Fisher’s exact test (FIG.8D), two-tailed unpaired Wilcoxon’s tests (boxplot in FIGs.8F, 8G, 8H), and K-S test (cumulative plot in FIG. 10 8F). All presented data were from HEK293T cells.
  • FIGs.9A-9H provide plots, graphs, a drawing, a diagram, and a pie chart showing that protein members of the Box C/D snoRNP tend to show up-regulated RNA expression in cancer samples relative to control samples and bind mRNAs in C4-2 cells. Boxplot showing RNA 15 expression of the snoRNP proteins FBL (FIG.9A), NOP56 (FIG.9B), and NOP58 (FIG.9C) in cancer and normal patient samples of individual cancer types in the TCGA dataset.
  • FIG.9D is a flowchart of RIP-seq experiment to detect FBL-binding RNAs.
  • FIG.9E is a Venn diagram showing the overlap of FBL-binding mRNAs between C4-2 and HEK293T cell lines.
  • FIG.9G shows that the IGV genome browser shows examples of C/D box snoRNAs bound by FBL in C4-2 cells.
  • FIG.9H shows that the IGV genome browser shows examples of mRNAs bound by FBL in C4-2 cells.
  • P value was calculated by Fisher’s exact test (FIG.9E).25
  • FIG.10A-10G provide a flowchart, plots, graphs, and piecharts showing a machine- learning strategy to detect Nm modification based on Nanopore direct RNA-seq.
  • FIG.10A shows the computational pipeline for detecting RNA Nm sites from Nanopore directive RNA- seq.
  • FIGs.10B, 10C are bar plots showing the accuracy (FIG.10B) and AUROC (FIG.10C) of the machine learning model when applied to each 1005-mers in KTC-1 cells.
  • FIG.10D is a bar30 plot showing RNA expression level of FBL determined by Nanopore RNA-seq in individual C4- 2 samples.
  • FIG.10F is a nested pie chart to show percentages of A, U, G, and C bases among detected Nm sites in C4-2 cells (outer), all bases in mRNAs (middle), and bases in the 5-mers covered by the machine-learning model (inner). The sections of the chart are as 5 follows, from the top moving clockwise: U; C; A; G.
  • FIGs.12T-12U are bar plots showing the overlap between Nm sites detected 30 by our method and the Nm-mut-seq method in HeLa (FIG.12T) and HepG2 (FIG.12U) cells.
  • FIG.12V is a bar plot showing the overlap of Nm sites detected by our method and sites of several other modification types in HEK293T cells. The left bar in each set of two is Overlap, and the right bar is Expected.
  • FIG.12W is a scatter plot showing expression correlation between FBL and PSMD13 in the GTEx database. 86 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • FIGs.13K-13U are box plots to show 3′ UTR length (FIG.13K), number of potential miRNA-binding motifs in 3′ UTR (FIG.13L), 3′ UTR ARE score (FIG.13M), 5′ UTR length (FIG.13N), number of miRNA-binding motifs in 5′ UTR (FIG.13O), CDS length (FIG.13P), number of miRNA-binding motifs in CDS (FIG.13Q), 15 intron length (FIG.13R), 5′ UTR GC content (FIG.13S), 3′ UTR GC content (FIG.13T), and CDS GC content (FIG.13U) of individual gene groups.
  • FIGs.13V-13X show Length of the 3′ UTR (FIG.13V), mRNA half-life (FIG.13W) with Nm only happened in CDS and only occurred in 3′ UTR in C4-2 cells. P values were determined 20 by a two-tailed unpaired Wilcoxon test (FIGs.13A-13H, 13K-13W).
  • FIGs.14A-14E provide graphs and plots showing RNA-binding proteins with a potential to mediate Nm function.
  • FIG.14A shows distributions of the protein-binding Nm sites in different sequence regions of mRNA in HEK293T cells. For each bar, the sections of the bar are divided, from left to right, as follows: 3’-UTR; Exon; Non-Coding; Promoter; 5’-UTR; Intron. 25
  • FIG.14B shows enrichment of the binding regions for individual RNA-binding proteins around Nm sites in HEK293T cells. FBL and TNRC6C were shown as the positive and negative controls.
  • FIG.14C is a boxplot showing mRNA half-life of CPSF7-binding transcripts under FBL knockdown and control conditions in HEK293T cells.
  • FIGs.14D-14E show Genome browser tracks showing RNA- 30 seq read density at the 3′ UTR (FIG.14D) or gene body (FIG.14E) of each gene in HepG2 cells.
  • the disclosure features compositions comprising non-naturally occurring RNAs modified 5 at internal sites and methods for stabilizing RNA (e.g., mRNA).
  • the disclosure is based, at least in part, on the discovery, described herein, that modification of RNA through 2’-O-methylation (Nm or 2'-O-Me) at internal sites within the RNA surprisingly increased the stability of the RNA.
  • the RNA is an mRNA modified to include Nm modifications at internal sites, at least in part through the 10 activity of fibrillarin (FBL).
  • the RNA is in vitro transcribed or chemically synthesized and then 2’-O-methyl modified.
  • the in vitro transcribed or chemically synthesized RNA comprises an increased number of Nm modifications relative to a naturally occurring RNA, or comprises Nm modifications at residues that are not modified in a naturally occurring RNA.
  • the in vitro transcribed or 15 chemically synthesized RNA is an mRNA, rRNA, tRNA, miRNA, snRNA, snoRNA, viral RNA, or RNA/DNA hybrid that comprises one or more Nm modifications.
  • the RNA is an mRNA comprising Nm modifications at 10%, 20%, 50%, 75% or 100% of ribonucleosides (e.g., internal sites).
  • Nm modification and the binding of FBL 20 on mRNA stability in both FBL- and NOP56-deficient cell lines was investigated, as compared to control cells.
  • Nm A connection 30 between Nm and sequence-encoded features was observed, including 3′ UTR length, ARE (AU rich elements) score, miRNA binding sites, and GC content. For instance, it was found that Nm connected to alternative polyadenylation, which leads to 3′ UTR shortening and thus increased mRNA stability.
  • RNA modifications play pivotal roles in regulating RNA processing and translation (Roundtree et al. (2017), Cell 169, 1187-1200). Detecting RNA modifications at individual sites on each RNA molecule in the cell is a key step to understanding the function and regulation of these modifications. To date, however, the function of the Nm at mRNA internal sites in the 10 cells, e.g., the effect on mRNA stability and the underlying mechanism, is unclear.
  • 2’-O-methylation A particular type of RNA modification, 2'-O-methylation (2'-O-Me or Nm), stands out due to its widespread occurrence on all four types of nucleotides (A, U, G, C) and in most RNA 15 categories, e.g., mRNA, rRNA, tRNA, miRNA, snRNA, snoRNA, and viral RNA. Modified RNAs described herein include all of these categories of RNAs. Chemically, 2'-O-Me consists of a methyl group being added to the oxygen atom that is attached to the second carbon in the ribose sugar of a nucleotide (FIG.1A).
  • the most prevalent site for 2'-O-Me in mRNA is the mRNA 5’ cap.
  • This cap is a20 protective structure found at the 5’ end of eukaryotic mRNA and plays an important role in pre- processing and stabilizing mRNA.
  • the 2'-O-Me modification on the first nucleotide in the mRNA cap known as N12'-O-Me (Cap1), is specifically catalyzed by the enzyme cap methyltransferase 1 (CMTR1) that adds a methyl group onto the 2'-O-ribose of the first transcribed nucleotide.
  • CMTR2 is responsible for ribose 2'-O-Me at the second 25 transcribed nucleotide (Cap2).
  • FBL C/D-box small nucleolar RNAs guide RNA methyltransferase Fibrillarin (FBL), which forms a complex with other proteins such as the 89 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 NOP56, NOP58, and SNU13, to catalyze Nm on rRNA.
  • FBL plays essential roles in many biological processes such as histone H2AQ104 methylation and eye and craniofacial development.
  • Nm-seq (Dai et al., Nat Methods 14, 695-698) and Nm-Mut-seq (Chen et al., Cell Res 33, 727-730) enabled transcriptome-wide detection of Nm sites and successfully revealed that Nm modifications were present at internal sites of mRNA.
  • a quantitative way to directly measure the abundance of transcriptome-wide Nm modification with stoichiometric information 15 is still lacking.
  • Nanopore direct RNA-seq represents a promising approach to concurrently detect multiple types of RNA modifications based on electrical signals of the nucleotides (Garalde, et al. (2016).
  • Polynucleotides of the disclosure comprise modified ribonucleotides comprising a 2’-O- methyl modification that increases the stability of the polynucleotide.
  • the polynucleotide is an RNA molecule where at least about 10% of the internal sites (i.e., sites between the 5’ and 3’ termini comprise a 2’-O-methyl modification).
  • RNA is a modified mRNA.
  • the RNA is chemically synthesized or in vitro transcribed.
  • Modified mRNA refers to a nucleic acid molecule comprising at least one modified nucleoside.
  • a modified mRNA is generated by in vitro transcription or 5 chemical synthesis.
  • RNA polymerase is a phage RNA polymerase.
  • the modified nucleotides pseudouridine, m5U, s2U, m6A, and m5C are known to be compatible with transcription using phage RNA polymerases.
  • Polymerases that accept modified nucleosides are known to those of skill in the art. 25 It is also contemplated that modified polymerases can be used to generate modified mRNAs, as described herein.
  • a polymerase that tolerates or accepts a particular modified nucleoside as a substrate can be used to generate a synthetic, modified mRNA including that modified nucleoside.
  • aa modified mRNA is translatable in vivo by the translation 30 machinery of a eukaryotic, preferably mammalian, and more preferably, human cell. Translation generally requires at least a ribosome binding site, a methionine start codon, and an open reading frame encoding a polypeptide.
  • the modified RNA RNA is an mRNA that comprises a 5' cap, a stop codon, a Kozak sequence, and/or a polyA tail.
  • the RNA is a non-coding mRNA. In still other embodiments, the RNA is a microRNA. 91 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Some nucleoside modifications alter the rate of translation.
  • fluoro-modified nucleosides are generally not translatable and can be used herein as a negative control for an in 10 vitro translation assay.
  • the modified mRNA has an increased half-life, and/or shows increased resistance to degradation through enzymatic or other regulatory means (e.g., in vivo, such as in a cell), as compared to an unmodified mRNA.
  • Methods for Increasing mRNA Stability The present disclosure provides methods of increasing the stability of RNA molecules (e.g., mRNAs). These methods involve modifying the mRNA to create modified mRNA.
  • mRNA stability refers to mRNA having an increased half-life, and/or showing increased resistance to degradation through enzymatic or other regulatory means (e.g., in vivo, 20 such as in a cell), as compared to an unmodified mRNA. Accordingly, the methods herein modify mRNAs as described herein to include modifications to prevent rapid degradation by endo- and exo-nucleases, or otherwise increase half-life, as compared to unmodified versions of these mRNAs. In some embodiments, the mRNA is modified to include a nucleoside having a 2’-O-methyl modification.
  • the 2’-O-methyltransferase polypeptide is selected from the group consisting of cap methyltransferase 1 (CMTR1), cap30 methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O- methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA 92 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF.
  • CMTR1 cap
  • the method involves identifying one or more miRNA binding sites within an mRNA and targeting the identified miRNA sites for modification using a 2’-O-methyltransferase.
  • 2’-O-methyltransferases useful in 5 embodiments of the present disclosure are discussed in more detail, for example, in Dimitrova et al., Genes (2019) 10:117, the contents of which are incorporated herein by reference.
  • mRNAs including 2’-O-methyl modifications to nucleosides at internal sites within the mRNA are created using methods such as in-vitro transcription.
  • In-vitro transcription to produce modified mRNAs of the present disclosure may be performed using 10 RNA polymerases.
  • the RNA polymerases are T7 polymerases.
  • the RNA polymerases include thermostabilizing mutations.
  • thermostabilized mutants of T7 RNA polymerase include one or more mutations selected from the group consisting of: R425 (e.g., R425C), G542 (e.g., G542V), Y639 (e.g., Y639G, Y639F, Y639V), H784 (e.g., H784A, H784G, H784S), E593G, V685A, I119V, G225S, 15 K333N, D366N, F400L, S661G, F880Y, H772R, S430P, N433T, S633P, F849I, P266L.
  • R425 e.g., R425C
  • G542 e.g., G542V
  • Y639 e.g., Y639G, Y639F, Y639V
  • H784 e.g., H784A, H
  • Mutant T7 RNA polymerases useful in embodiments of the present disclosure are described further, for example, in Meyer et al., Nucleic Acids Research (2015) 43(15):7480-7488, the contents of which are hereby incorporated by reference.
  • the use of in-vitro transcription to create modified RNAs including 2’-O-methyl modifications at internal sites is described further, for example, in 20 Cavayer et al., Nature (1996) 383:732-735, Zhao and Yu, Nature Methods (2008) 5(1):95-100, Ge et al., RNA (2010) 16:1078-1085, and Elliott et al., Nature Communications (2019) 10:3401, the contents of each of which are hereby incorporated by reference.
  • the modified mRNAs described herein can further comprise a 5' and/or 3' untranslated region (UTR).
  • Untranslated regions are regions of the RNA before the start codon (5') and after the stop codon (3'), and are therefore not translated by the translation machinery. Modification of an RNA molecule with one or more untranslated regions can improve the stability of an mRNA, 15 since the untranslated regions can interfere with ribonucleases and other proteins involved in RNA degradation. In addition, modification of an RNA with a 5' and/or 3' untranslated region can enhance translational efficiency by binding proteins that alter ribosome binding to an mRNA.
  • Modification of an RNA with a 3' UTR can be used to maintain a cytoplasmic localization of the RNA, permitting translation to occur in the cytoplasm of the cell.
  • the modified mRNAs described herein do not comprise a 5' or 3' UTR.
  • the modified mRNAs comprise either a 5' or 3' UTR.
  • the modified mRNAs described herein comprise both a 5' and a 3'UTR.
  • the 5' and/or 3' UTR is selected from an mRNA known to have high stability in the cell (e.g., a murine alpha-globin 3' UTR).
  • the 5' UTR, the 3' UTR, or both comprise one or 25 more modified nucleosides.
  • the modified mRNAs described herein further comprise a Kozak sequence.
  • the "Kozak sequence” refers to a sequence on eukaryotic mRNA having the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ⁇ G ⁇ .
  • the Kozak consensus sequence is 30 recognized by the ribosome to initiate translation of a polypeptide. Typically, initiation occurs at the first AUG codon encountered by the translation machinery that is proximal to the 5' end of the transcript.
  • the modified mRNAs described herein further comprise a Kozak consensus sequence at the desired site for initiation of translation to produce the correct length polypeptide.
  • the Kozak sequence comprises one or more modified nucleosides.
  • the modified mRNAs described herein further comprise a "poly (A) tail", which refers to a 3' homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides).
  • the inclusion of a 3' poly(A) tail can protect the modified mRNA from degradation 10 in the cell, and also facilitates extra-nuclear localization to enhance translation efficiency.
  • the poly(A) tail comprises between 1 and 500 adenine nucleotides; in other embodiments the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 15 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 adenine nucleotides or more.
  • the poly(A) tail comprises between 1 and 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises between 90 and 120 adenine nucleotides. In some such embodiments, the poly(A) tail comprises one or more modified nucleosides. 20 It is contemplated that one or more modifications to the modified mRNAs described herein permit greater stability of the modified mRNA in a cell or tissue in vivo. To the extent that such modifications permit translation and either reduce or do not exacerbate a cell's innate immune or interferon response to the modified mRNA with the modification, such modifications are specifically contemplated for use herein.
  • a modified mRNA as described herein does not comprise an AU-rich region.
  • the 3' UTR substantially lacks AUUUA sequence elements. Oligonucleotide Synthesis and Purification
  • the modified mRNA can be chemically synthesized using methods described herein.
  • RNA may be produced enzymatically or by partial/total organic 99 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis.
  • each strand is prepared chemically. Methods of synthesizing RNA molecules are known in the art, in particular, the chemical synthesis methods as described in Verma and Eckstein (1998) or as described herein.
  • modified mRNA molecules can 5 by synthesized using solid phase oligonucleotide synthesis methods as described in, for example, Usman et al., U.S. Pat. Nos.5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086.
  • modified mRNA molecules comprising one or more modified nucleotides are synthesized, using solid phase phosphoramidite chemistry, deprotected and 10 desalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie, 1993; Wincott et al., 1995).
  • the oligomers may be purified using ion-exchange high performance liquid chromatography (IE-HPLC), for example, on an Amersham Source 15Q column (1.0 cm.times.25 cm) (Amersham Pharmacia Biotech, Piscataway, N.J.) using a step-linear gradient.
  • IE-HPLC ion-exchange high performance liquid chromatography
  • Example 1 Nm at mRNA internal sites is positively associated with mRNA stability 5
  • ActD Actinomycin D
  • GSEA Gene Set Enrichment Analysis
  • FBL is one of the enzymes well known to catalyze Nm and NOP56 is 15 required to facilitate the reaction
  • new RNA-Seq was performed following ActD inhibition of transcription to profile RNA half-life in control, FBL-knockdown and NOP56-knockdown HEK293T cells (FIGs.1D, 8A-8C).
  • the mRNAs that were 2′-O-methylated in the control cells were significantly more likely to show a decrease of mRNA half-life in response to the knockdown of FBL or20 NOP56 (FIGs.1E-1F).
  • the human PCa cell line C4-2 was a gift from Dr. Leland W. Chung at Cedars-Sinai Medical Center and was cultured in RPMI 1640 medium supplemented with 10% fetal bovine 5 serum (FBS).
  • FBS fetal bovine 5 serum
  • HEK293T cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% FBS.
  • the PCR mixture contains 10 ⁇ L amfisure PCR Master Mix (GenDEPOT), 2 ⁇ L cDNA and 10 mM PCR primers.
  • the PCR products were then separated on 1% agarose gels and visualized by a Bio-rad imaging system. DNA band signal intensities were analyzed using ImageJ software. The methylation ratio of each group was determined from the 15 densities of PCR bands obtained using high and low dNTP concentrations.
  • Total RNA extraction and quality assessment Total RNA was extracted using RNAprep Pure Kit (polysaccharides and polyphenolics- rich) and treated with DNase I to remove DNA. Firstly, the quality of total RNAs was detected 20 using 1% agarose electrophoresis, and no RNA degradation was observed.
  • RNA half-life of the HEK293T cell and C4-2 cells was calculated as described before (Li, et al. (2023). Nucleic Acids Res 51, 6020-6038. 10.1093/nar/gkad300). The RNA levels were first normalized to RPKM (Reads Per Kilobase Million). To reduce the noise due to genes that showed a higher expression level after inhibition of transcription, the RPKM values were scaled based on the expression levels of the top 10 genes 115 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO.
  • control or 10 FBL-deficient C4-2 cells were cultivated in 1640 medium containing 100 ⁇ M 4-thiouridine (S4U) for a continuous 24-hour duration. Subsequently, the S4U-containing medium was exchanged by medium with an excess (10 mM) of unmodified uridine (U), and cell samples were collected at specific time points. The reference point for these time points was set at 0 hour, corresponding to the moment of S4U removal. RNA isolation was performed on all collected 15 samples by Trizol (Thermo), and the 4-thiol groups present on S4U-labeled transcripts were alkylated with iodoacteamide.
  • S4U 4-thiouridine
  • the libraries were generated from 70-200 ng of input RNA using QuantSeq 3’ mRNA-Seq Library Prep Kit (LEXOGEN), followed by shipment to Admera Health for sequencing. To estimate mRNA half-lives, a published SLAM-seq protocol was followed. Initially, 20 raw reads in fastq files underwent quality checking and trimming using fastp (Chen, et al., (2016). Bioinformatics 34, i884-i890.10.1093/bioinformatics/bty560). Subsequently, these reads were processed with slamdunk (Neumann et al., (2019). BMC Bioinformatics 20, 258. 10.1186/s12859-019-2849-7).
  • CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 (Promega) was added to each well.
  • the plates were then placed on an orbital shaker at 37°C for 10 minutes to induce cell lysis, and bioluminescence was measured using a Tecan plate reader.
  • Wound healing assay 5 Control or PSMD13-deficient C4-2 cells were cultured in 35 mm dishes with 3-well Culture-Insert (Ibidi) till grown to 80% confluency. After carefully removing the inserts, the dishes were replenished with serum-free 1640 medium and sustained in a humidified environment at 37°C. Microscopic images were captured at the 0-hour and 30-hour marks, and the extent of cell migration was quantified by ImageJ software.
  • the mRNA concentration was measured by Qubit RNA HS assay kit (ThermoFisher, Q32855), and the quality was confirmed by Agilent Bioanalyzer RNA Pico assay chip. Then, the mRNA was subjected to library construction using an Oxford Nanopore direct RNA sequencing kit (SQK-RNA002) according to the manufacturer’s manual. 30 500 ng of mRNA was first annealed, ligated to RT adapter, and then reverse-transcribed to form DNA-RNA hybrid products. Then, the RNA adapter was ligated to the products and was ready for sequencer loading.
  • Training features were extracted, including the median, standard deviation, mean, and dwell time of the Nanopore signal for the k-mer around each Nm site in the rRNA. On the other hand, these features of the same k-mer were also extracted from the IVT reads of mRNA. Then, an XGBoost model was trained by dividing the 15 features into the training and testing sets with a ratio of 4:1 for cross-validation. The XGBoost model was constructed using the machine learning library Scikit-learn. The receiver operating characteristic (ROC) curve and the area under the ROC (AUROC) were used to evaluate the performance of the model.
  • ROC receiver operating characteristic
  • AUROC area under the ROC
  • a base found to be modified in at least 5 transcripts was identified as a modified Nm site.
  • Differentially methylated sites between normal and knockdown cells were calculated by the R package REDITs (https://github.com/gxiaolab/REDITs) (Tran et al., (2020). 30 Bioinformatics 36, 2796-2804.10.1093/bioinformatics/btaa066).
  • the average density of the Nm site across the transcript body was plotted using the Guitar R/Bioconductor package (Cui (2016). Biomed Res Int 2016, 8367534.10.1155/2017/8367534).
  • a similar method is applied to the Nm detection of rRNA in yeast.
  • Nanopore direct RNA-seq data of rRNA in WT, snR60, snR61, and snR62 strains of yeast (Begik et al., Nat Biotechnol 39, 1278-1291), human HepG2 and 119 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 HeLa cell (Tavakoli, et al., (2023). Nat Commun 14, 334.10.1038/s41467-023-35858-w), and Drosophila cells (Sklias et al., (2023).
  • the RIP experiment in C4-2 cells was performed using the EZ-Magna RIP kit (Millipore), following the procedure provided by the manufacturer.
  • C4-2 cells were lysed in RIP lysis buffer and incubated with FBL antibody-magnetic beads mixture at 4 °C overnight to enrich RNA binding protein.
  • the RNA that bound to FBL protein was collected by Proteinase K digestion, followed by purification and precipitation.
  • input and RIP-ed RNA samples were shipped to BGI for library preparation and sequencing using the DNBseq platform. After mapping to the genome hg38 using tophat (version 2.1.1) with default parameter values and UCSC known genes as a reference, the software piranha (Uren et al., (2012).
  • Bioinformatics 28, 3013-3020. 10.1093/bioinformatics/bts569) was used to analyze the RIP-seq data with default parameters.
  • the Homer annotatePeaks.pl script was used to annotate peaks from the RIP-seq data (Heinz et al., (2010). Mol Cell 38, 576-589.10.1016/j.molcel.2010.05.004).
  • a P-value cutoff of 0.01 was used to define FBL-binding RNAs in the C4-2 cells.
  • RNA immunoprecipitation (RIP)-qPCR 25 The RIP experiment was conducted using the EZ-Magna RIP kit (Millipore) following the User’s Guide.
  • control or FBL-deficient C4-2 cells were lysed in RIP lysis buffer and incubated with anti-CPSF7 antibody and protein A/G magnetic beads mixture at 4 °C overnight to enrich the RNA-bound CPSF7 proteins.
  • the CPSF7-associated RNAs were extracted by Proteinase K digestion, followed by purification and reverse transcription into cDNA. Then, the 30 qPCR assay was performed to measure the %Input of selected mRNA candidates in each group using the primers shown in Supplementary Table 6.
  • Functional enrichment analysis of gene groups KEGG functional enrichment analysis was performed using the R package clusterProfiler (v4.0.5) (Wu, et al. (2021).
  • clusterProfiler 4.0 A universal enrichment tool for interpreting omics data. Innovation (Camb) 2, 100141.10.1016/j.xinn.2021.100141).
  • Gene Set Enrichment 5 Analysis was performed using the GSEA Java software (v4.0.3) (Subramanian et al., (2005). Proc Natl Acad Sci U S A 102, 15545-15550.10.1073/pnas.0506580102).
  • PLoS Genet 8, e1002433.10.1371/journal.pgen.1002433) and miRNA 15 binding sites of UTRs and CDS from the miRWalk database (Sticht et al., (2016). PLoS One 13, e0206239.10.1371/journal.pone.0206239) were downloaded. Dapars2 was used to perform APA analysis with the default parameters (Xia et al., (2014). Nat Commun 5, 5274. 10.1038/ncomms6274; Li, et al. (2021). Nat Genet 53, 994-1005.10.1038/s41588-021-00864-5).
  • a gene was considered to be regulated by FBL if its PDUI values from both replicates were 20 lower or greater in FBL-knockdown cells than in the control cells and if the difference in average PDUI values was larger than 0.1.
  • RNA binding proteins co-localized with Nm CLIP-seq database of RNA binding proteins in HEK293/HEK293T cells were retrieved 25 from the POSTAR3 database (Zhao et al., Nucleic Acids Res 50, D287-D294), and the overlapped regions of each file were merged using “bedtools merge”. These regions were overlapped with Nm sites in HEK293T cells.

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Abstract

Provided herein are methods for modifying mRNA, thereby stabilizing the mRNA. In particular, polypeptides of the disclosure may be used to modify mRNA with 2'-O-methylations.

Description

ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 COMPOSITIONS AND METHODS FOR STABILIZING mRNA 5 CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority to and the benefit of U.S. App. No.63/575,132, filed April 5, 2024, the contents of which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION 10 RNA-based therapeutics are a promising new field of therapy. However, RNA is inherently unstable, especially in vivo. With the growing number of RNA therapeutics and RNA vaccines approved for clinical uses, RNA has attracted exponentially growing interest of both academic researchers and pharmaceutical industry. Accordingly, there is a need for mRNA 15 having increased stability. SUMMARY OF THE INVENTION The present disclosure provides compositions comprising modified RNA having 20 increased stability, methods for stabilizing RNA (e.g., mRNA), and methods of using modified RNA having increased stability. Compositions and articles defined by the disclosure were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims. 25 In an aspect, the present disclosure provides an in vivo method of stabilizing an mRNA. The method involves contacting an mRNA with a heterologous 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA. In another aspect, the present disclosure provides an in vitro method of stabilizing an 30 mRNA. The method involves contacting an mRNA with a 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA. In another aspect, the present disclosure provides a method of stabilizing an mRNA. The method involves contacting an mRNA with a 2’-O-methyltransferase polypeptide, such that an 35 internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA, where the 2’-O-methyltransferase polypeptide includes one or more of: cap 1 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 methyltransferase 1 (CMTR1), cap methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA 5 methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA methyltransferase 11 homolog (TRMT11), tRNA methyltransferase 13 homolog (TRMT13), or tRNA methyltransferase 44 homolog (TRMT44). In another aspect, the present disclosure provides a method of producing a stabilized mRNA. The method involves using in-vitro transcription to produce a modified mRNA 10 including one or more 2’-O-methyl modifications at an internal site of the modified mRNA, In another aspect, the present disclosure provides an in vitro transcribed or chemically synthesized single stranded polynucleotide including one or more modified ribonucleotides including a 2’-O-methyl modification. In another aspect, the present disclosure provides an in vitro transcribed or chemically 15 synthesized single stranded polynucleotide including one or more modified ribonucleotides including a 2’-O-methyl modification at a non-naturally occurring position. In any of the above aspects, or embodiments thereof, the method is performed in-vitro or in-vivo. In any of the above aspects, or embodiments thereof, the 2’-O-methyltransferase is 20 heterologous. In any of the above aspects, or embodiments thereof, the 2’-O-methyltransferase includes a fibrillarin (FBL) polypeptide. In any of the above aspects, or embodiments thereof, the 2’-O- methyltransferase includes a FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3) polypeptide. In any of the above aspects, or embodiments thereof, the 2’-O-methyltransferase includes a 27S pre-rRNA 25 (guanosine2922-2'-O)-methyltransferase (Spb1) polypeptide. In any of the above aspects, or embodiments thereof, the 2’-O-methyltransferase polypeptide includes a polypeptide where the polypeptide includes one or more of: cap methyltransferase 1 (CMTR1), cap methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 30 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA methyltransferase 11 homolog (TRMT11), tRNA methyltransferase 13 homolog (TRMT13), or tRNA methyltransferase 44 homolog (TRMT44). 2 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 In any of the above aspects, or embodiments thereof, the modified mRNA is modified in comparison to a reference mRNA, wherein the reference mRNA is an unmodified or wild type version of the modified mRNA. In any of the above aspects, or embodiments thereof, method further involves 5 complexing the 2’-O-methyltransferase with a polynucleotide prior to contacting the mRNA with the 2’-O-methyltransferase, where the polynucleotide is at least partially complementary and/or capable of hybridizing to a target site within the mRNA. In any of the above aspects, or embodiments thereof, the polynucleotide is capable of directing the 2’-O-methyltransferase to the target site within the mRNA. 10 In any of the above aspects, or embodiments thereof, the method further involves identifying one or more microRNA (miRNA) binding sites within the mRNA prior to complexing the 2’-O-methyltransferase with a polynucleotide, where the polynucleotide is at least partially complementary and/or capable of hybridizing to one or more of the identified miRNA binding sites. 15 In any of the above aspects, or embodiments thereof, the in-vitro transcription is performed using an RNA polymerase. In any of the above aspects, or embodiments thereof, the RNA polymerase is a T7 RNA polymerase. In any of the above aspects, or embodiments thereof, the RNA polymerase includes one or more thermostabilizing mutations. In any of the above aspects, or embodiments thereof, the thermostabilizing mutations include one or more of: 20 R425X, R425C, G542X, G542V, Y639X, Y639G, Y639F, Y639V, H784X, H784A, H784G, H784S, E593G, V685A, I119V, G225S, K333N, D366N, F400L, S661G, F880Y, H772R, S430P, N433T, S633P, F849I, or P266L, where X is any amino acid. In any of the above aspects, or embodiments thereof, the modified mRNA is modified in comparison to a reference mRNA, where the reference mRNA is an unmodified or wild type 25 version of the modified mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of about 10% to about 300% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of about 50% to about 300% as compared to a 30 reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of about 50% to about 250% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of about 50% to about 200% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is 3 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 characterized as having an increased half life of at least about 10%, at least about 20%, at least about 30%, or at least about 40% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 50% as compared to a reference mRNA. In any of the above aspects, or 5 embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 70% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 90% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at 10 least about 100% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 150% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 200% as compared to a reference mRNA. In any of the above aspects, or 15 embodiments thereof, the modified mRNA is characterized as having an increased half life of at least about 250% as compared to a reference mRNA. In any of the above aspects, or embodiments thereof, the polynucleotide is an RNA molecule or a DNA/RNA hybrid. In any of the above aspects, or embodiments thereof, at least about 10% of the 20 ribonucleotides include a 2’-O-methyl modification. In any of the above aspects, or embodiments thereof, at least about 15% of the ribonucleotides include a 2’-O-methyl modification. In any of the above aspects, or embodiments thereof, at least about 20% of the ribonucleotides include a 2’-O-methyl modification. In any of the above aspects, or embodiments thereof, at least about 25% of the ribonucleotides include a 2’-O-methyl 25 modification. Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The 30 following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The 4 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. By “2’-O-methyltransferase” is meant an enzyme capable of modifying an RNA to comprise a 2’-O-methyl modification. In some embodiments, the RNA is an mRNA. In some 5 embodiments, the 2’-O-methyltransferase is one or more of cap methyltransferase 1 (CMTR1), cap methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O- methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA 10 methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA methyltransferase 11 homolog (TRMT11), tRNA methyltransferase 13 homolog (TRMT13), or tRNA methyltransferase 44 homolog (TRMT44). By “agent” is meant a polypeptide, nucleic acid molecule, or small compound. In embodiments, the agent is a modified RNA (e.g., RNA comprising a 2’-O methyl modification). 15 By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or condition. By "alteration" is meant a change (increase or decrease). As used herein, an alteration includes a 10% change, a 25% change, a 40% change, or a 50% or greater change in levels of an analyte. In embodiments, the alteration is the number or percent of nucleobases featuring a 2’ O- 20 methyl modification. In other embodiments, the alteration is an increase in the stability of a modified polynucleotide (e.g., 2x, 3x, 4x, 5x, 10x, 100x) relative to the stability of an unmodified polynucleoide (e.g., RNA) By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a 25 corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. 30 By “cap methyltransferase 1 polypeptide” or “CMTR1 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_055865.1, or a fragment thereof, and having methyltransferase activity. The sequence of an exemplary CMTR1 polypeptide is provided below: 5 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_055865.1 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 [Homo sapiens] MKRRTDPECTAPIKKQKKRVAELALSLSSTSDDEPPSSVSHGAKASTTSLSGSDSETEGKQHSSDSFDDA FKADSLVEGTSSRYSMYNSVSQKLMAKMGFREGEGLGKYSQGRKDIVEASSQKGRRGLGLTLRGFDQELN 5 VDWRDEPEPSACEQVSWFPECTTEIPDTQEMSDWMVVGKRKMIIEDETEFCGEELLHSVLQCKSVFDVLD GEEMRRARTRANPYEMIRGVFFLNRAAMKMANMDFVFDRMFTNPRDSYGKPLVKDREAELLYFADVCAGP GGFSEYVLWRKKWHAKGFGMTLKGPNDFKLEDFYSASSELFEPYYGEGGIDGDGDITRPENISAFRNFVL DNTDRKGVHFLMADGGFSVEGQENLQEILSKQLLLCQFLMALSIVRTGGHFICKTFDLFTPFSVGLVYLL YCCFERVCLFKPITSRPANSERYVVCKGLKVGIDDVRDYLFAVNIKLNQLRNTDSDVNLVVPLEVIKGDH 10 EFTDYMIRSNESHCSLQIKALAKIHAFVQDTTLSEPRQAEIRKECLRLWGIPDQARVAPSSSDPKSKFFE LIQGTEIDIFSYKPTLLTSKTLEKIRPVFDYRCMVSGSEQKFLIGLGKSQIYTWDGRQSDRWIKLDLKTE LPRDTLLSVEIVHELKGEGKAQRKISAIHILDVLVLNGTDVREQHFNQRIQLAEKFVKAVSKPSRPDMNP IRVKEVYRLEEMEKIFVRLEMKIIKGSSGTPKLSYTGRDDRHFVPMGLYIVRTVNEPWTMGFSKSFKKKF FYNKKTKDSTFDLPADSIAPFHICYYGRLFWEWGDGIRVHDSQKPQDQDKLSKEDVLSFIQMHRA 15 By “cap methyltransferase 1 polynucleotide” or “CMTR1 polynucleotide” is meant a nucleic acid molecule encoding an CMTR1 polypeptide or fragment thereof. The sequence of an exemplary CMTR1 polynucleotide is provided below: >NM_015050.3 Homo sapiens cap methyltransferase 1 (CMTR1), mRNA 20 GCAGTACTGGACCCGAGCGCGACGGTGCGGCTGGCGGACCCGGGCTGGCTTGTGGGGAAACGAAACTGAG GGAGGAGGCGGCGGCTCTGGCAGCGGCGGCGACAGTGTCGGCCTGACCCCCCCTCCGCTCCCCGGCAGCT CGCTCTCTCCCCTCAGCTTAACGATGAAGAGGAGAACTGACCCAGAATGCACTGCCCCCATCAAGAAACA GAAAAAAAGAGTTGCAGAGCTTGCCCTGAGCCTCAGCTCCACGTCCGATGATGAACCTCCCTCCTCTGTC AGTCATGGAGCAAAAGCATCTACTACAAGCCTTAGTGGGTCTGATAGTGAGACCGAGGGGAAACAACACA 25 GCTCTGACTCTTTTGACGATGCATTCAAAGCAGACTCTCTTGTGGAAGGAACTTCTTCTCGCTATTCCAT GTATAATAGCGTCTCCCAGAAGCTTATGGCCAAGATGGGCTTCAGGGAAGGTGAAGGATTGGGTAAATAC AGCCAGGGTCGGAAGGACATCGTTGAGGCTTCCAGTCAGAAAGGTCGAAGAGGCTTGGGTCTGACACTCC GGGGCTTTGACCAGGAGCTGAACGTGGACTGGCGAGATGAGCCAGAGCCCAGTGCTTGTGAGCAGGTGTC ATGGTTTCCAGAATGTACCACTGAAATTCCTGACACTCAGGAAATGAGCGATTGGATGGTGGTGGGAAAG 30 AGAAAGATGATTATTGAAGATGAAACAGAGTTTTGTGGGGAAGAGCTGCTTCACAGTGTGTTGCAGTGTA AGAGCGTGTTTGATGTCTTGGATGGGGAAGAGATGCGGCGAGCTCGGACTCGGGCCAATCCCTATGAGAT GATCCGAGGAGTCTTCTTTCTAAACAGGGCAGCAATGAAGATGGCTAACATGGATTTTGTATTTGATCGC ATGTTCACAAATCCGCGGGACTCTTATGGGAAGCCACTGGTGAAGGACCGGGAAGCTGAGCTTCTGTACT TTGCTGATGTCTGCGCAGGCCCAGGTGGCTTCTCAGAGTATGTGCTGTGGAGGAAGAAGTGGCATGCAAA 35 GGGCTTTGGAATGACTTTGAAGGGCCCTAATGACTTCAAGCTGGAGGACTTCTACTCTGCTTCCAGTGAA CTCTTCGAACCCTACTATGGTGAGGGTGGGATTGATGGAGATGGAGATATCACCCGCCCAGAGAACATCT CTGCTTTTCGGAATTTTGTCCTGGATAACACAGATCGCAAGGGTGTCCATTTTCTGATGGCTGATGGGGG TTTCTCGGTGGAGGGGCAGGAGAACCTGCAGGAGATCCTCAGCAAGCAGCTGCTTCTGTGTCAGTTCCTC ATGGCGCTGTCCATTGTCCGGACAGGAGGCCACTTCATCTGTAAAACCTTTGACCTGTTCACACCGTTTA 40 GTGTGGGGCTTGTCTACCTGCTGTACTGCTGCTTTGAACGAGTTTGTCTCTTCAAGCCTATTACCAGCCG 6 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TCCTGCCAACTCAGAGAGGTATGTGGTGTGCAAGGGCCTGAAGGTGGGCATAGATGATGTTCGGGATTAC CTCTTCGCAGTGAATATTAAACTCAATCAGCTGCGGAACACGGATTCCGACGTCAACTTGGTGGTCCCCC TGGAGGTGATCAAGGGAGACCATGAATTTACTGACTACATGATACGGTCCAATGAGAGCCACTGTAGTCT GCAGATCAAAGCTCTGGCGAAAATCCATGCCTTTGTTCAAGACACGACACTGAGTGAGCCTCGACAGGCA 5 GAGATACGGAAGGAGTGCCTCCGACTCTGGGGGATCCCAGACCAGGCTCGTGTGGCTCCTTCTTCCTCCG ACCCTAAATCGAAGTTCTTTGAGCTAATCCAGGGCACTGAGATTGACATCTTCAGCTACAAGCCCACACT GCTCACCTCTAAAACCCTGGAGAAGATCCGCCCTGTGTTTGACTACCGCTGCATGGTATCTGGCAGTGAG CAGAAGTTCCTCATCGGCCTGGGGAAATCCCAGATCTACACATGGGATGGCCGCCAGTCAGACCGCTGGA TCAAGCTAGACCTGAAGACAGAGCTGCCCCGGGACACTCTGCTATCTGTGGAAATTGTGCATGAGCTGAA 10 AGGGGAGGGGAAGGCCCAGAGGAAGATCAGTGCCATCCACATCCTCGATGTCCTTGTGCTGAATGGCACC GACGTTCGGGAGCAGCACTTTAACCAGCGAATTCAGCTTGCCGAGAAATTTGTGAAAGCCGTTTCCAAGC CTAGTCGGCCCGACATGAATCCCATCAGGGTGAAGGAGGTGTACAGACTGGAAGAGATGGAGAAGATTTT TGTCAGGTTGGAGATGAAGATCATCAAGGGCTCCAGTGGCACCCCAAAGCTCAGCTACACAGGGCGTGAT GACCGGCACTTTGTACCCATGGGCCTCTACATCGTCAGGACAGTGAATGAGCCCTGGACTATGGGATTCA 15 GCAAAAGCTTCAAGAAGAAGTTCTTCTACAACAAGAAAACCAAGGACTCTACTTTTGACCTCCCTGCAGA CTCCATTGCCCCATTTCACATTTGCTACTATGGCCGGCTCTTCTGGGAGTGGGGGGATGGCATTCGTGTG CATGACTCCCAGAAGCCCCAGGACCAGGACAAGCTGTCCAAGGAGGACGTCCTCTCCTTCATCCAGATGC ACAGGGCCTAAGAGCCTCAGAATGTGCCACCCCTGCAGAATGCCCTGTCATTCCTGAGATGGGGCCACCT GGGGCCCACAGTGCTGGCTTCTTCCCCCTCTTGAAAAGGGACTGGGGAGCATTGCACCTGGCATGAGGAG 20 TGGGTGGCCTCCTCTCCATCCCCTGAAGAGCTCAGGCAGGGCCCTGCAGAGAACACTCATGTTCCTTCTG GGACACCTGCCTGGGAACTTTCCCCTGCCAGGACTCAGCCTGAAGGAAGCTGCTCCTGAGGCAGGTATGA GGTCAGTGCCTAGGGCACGTGGGACTGATGGAGGACATATCAGAGTGGCAGAGCTGTGGGCTCTGCTGTT CTCTCCTGCATCCTGTAGACTCACTTTTCTGAGTTCCATGCACTGCCCTGAGGGTAGCCATGCCCTTGCT TTGCCCAACTTTTTATTGGGCCATCCCTGAGTGGGTGGAGACCTGCTGTCATGAGCTGGCCAGGAGAACC 25 TGCTATAAAAAAATCAAGGTTTTGTTTCTTTGAACTTACTCTGTTTTGATGCCAAATTGGAGACCATTTT CTTGTCTCCTTCCCCCACTCATCCTGGCCTTCCCTGGAGTTCTTCCTAGCCCAGAGCTCTGACAGTCCAG CAGGGTGGGAAGGAGGGAGTTTGGGCAAACTCTCATCCCTGATACCACATTGAGATCCTGGGAGCCCTCT TTTCGTACTGAGTATGGAGTTGTAGAGCCATCCTAGGTGCCATCCCCTTTTGGTCCAAACATTGGGCAGC GCTAGATGGCAGGAAGCAGCCTTGAAGACCCGTCTTTCCCCCACAGCAGCAGGGGCCCCAGCAGTAACAA 30 AGGGTACCTCCAGGGGTTTGGGTAGCGCTGCCCTCTGGCAGTCATGCACCGCTGTCTGCCATAGCCGCTC TAGGGTCTTGGCAGAATTCTGAGCTTGAAGTGCAGCTCCCTTACTACCCTTTCCCTTCCTTTTTCTTCCC TAATAGGAGGTACAATCTGCTTTTGTTTGTCGTTAAGTGGTCACTCCCATTTCCTTTATCTTGGCCGACA ACACAGAGAGGAGGGGGAGCTGGGCAGTAGCTTGGGGTGGGGGTGGGCACCTGTGGTTGTTTTTAATGGG AAATACCTCTCAGAGATGTTCATGCAGGCTCCCTAGGGCCCCATCCCAGTGCCAGGCTGGTTTCCATGGA 35 GATAGGGCACTGAGGCTCCCGTGAGGTTGGAATCGACTTCACCATGGGGGTCCTTCAGCCAGCATCCAGC TCCCCACCCCCAGGCTGGCAGTAGCACTGCTGAGATGCTGTATTTCCACCCAATTCTGGGTATATCAGTG TGTCTTGCAGAATCTTGGATCATTAAAGATAAACATATTTTTAA By “cap methyltransferase 2 polypeptide” or “CMTR2 polypeptide” is meant a 40 polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. 7 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 NP_001093115.1, NP_001311303.1, NP_001311306.1, NP_001311307.1, NP_001311308.1, or NP_060818.4, or a fragment thereof, and having methyltransferase activity. The sequences of exemplary CMTR2 polypeptides are provided below: >NP_001093112.1 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2 5 [Homo sapiens] MSKCRKTPVQQLASPASFSPDILADIFELFAKNFSYGKPLNNEWQLPDPSEIFTCDHTELNAFLDLKNSL NEVKNLLSDKKLDEWHEHTAFTNKAGKIISHVRKSVNAELCTQAWCKFHEILCSFPLIPQEAFQNGKLNS LHLCEAPGAFIASLNHYLKSHRFPCHWSWVANTLNPYHEANDDLMMIMDDRLIANTLHWWYFGPDNTGDI MTLKFLTGLQNFISSMATVHLVTADGSFDCQGNPGEQEALVSSLHYCEVVTALTTLGNGGSFVLKMFTMF 10 EHCSINLMYLLNCCFDQVHVFKPATSKAGNSEVYVVCLHYKGREAIHPLLSKMTLNFGTEMKRKALFPHH VIPDSFLKRHEECCVFFHKYQLETISENIRLFECMGKAEQEKLNNLRDCAIQYFMQKFQLKHLSRNNWLV KKSSIGCSTNTKWFGQRNKYFKTYNERKMLEALSWKDKVAKGYFNSWAEEHGVYHPGQSSILEGTASNLE CHLWHILEGKKLPKVKCSPFCNGEILKTLNEAIEKSLGGAFNLDSKFRPKQQYSCSCHVFSEELIFSELC SLTECLQDEQVVVPSNQIKCLLVGFSTLRNIKMHIPLEVRLLESAELTTFSCSLLHDGDPTYQRLFLDCL 15 LHSLRELHTGDVMILPVLSCFTRFMAGLIFVLHSCFRFITFVCPTSSDPLRTCAVLLCVGYQDLPNPVFR YLQSVNELLSTLLNSDSPQQVLQFVPMEVLLKGALLDFLWDLNAAIAKRHLHFIIQREREEIINSLQLQN >NP_001311303.1 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2 [Homo sapiens] 20 MSKCRKTPVQQLASPASFSPDILADIFELFAKNFSYGKPLNNEWQLPDPSEIFTCDHTELNAFLDLKNSL NEVKNLLSDKKLDEWHEHTAFTNKAGKIISHVRKSVNAELCTQAWCKFHEILCSFPLIPQEAFQNGKLNS LHLCEAPGAFIASLNHYLKSHRFPCHWSWVANTLNPYHEANDDLMMIMDDRLIANTLHWWYFGPDNTGDI MTLKFLTGLQNFISSMATVHLVTADGSFDCQGNPGEQEALVSSLHYCEVVTALTTLGNGGSFVLKMFTMF EHCSINLMYLLNCCFDQVHVFKPATSKAGNSEVYVVCLHYKGREAIHPLLSKMTLNFGTEMKRKALFPHH 25 VIPDSFLKRHEECCVFFHKYQLETISENIRLFECMGKAEQEKLNNLRDCAIQYFMQKFQLKHLSRNNWLV KKSSIGCSTNTKWFGQRNKYFKTYNERKMLEALSWKDKVAKGYFNSWAEEHGVYHPGQSSILEGTASNLE CHLWHILEGKKLPKVKCSPFCNGEILKTLNEAIEKSLGGAFNLDSKFRPKQQYSCSCHVFSEELIFSELC SLTECLQDEQVVVPSNQIKCLLVGFSTLRNIKMHIPLEVRLLESAELTTFSCSLLHDGDPTYQRLFLDCL LHSLRELHTGDVMILPVLSCFTRFMAGLIFVLHSCFRFITFVCPTSSDPLRTCAVLLCVGYQDLPNPVFR 30 YLQSVNELLSTLLNSDSPQQVLQFVPMEVLLKGALLDFLWDLNAAIAKRHLHFIIQREREEIINSLQLQN >NP_001311306.1 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2 [Homo sapiens] MSKCRKTPVQQLASPASFSPDILADIFELFAKNFSYGKPLNNEWQLPDPSEIFTCDHTELNAFLDLKNSL 35 NEVKNLLSDKKLDEWHEHTAFTNKAGKIISHVRKSVNAELCTQAWCKFHEILCSFPLIPQEAFQNGKLNS LHLCEAPGAFIASLNHYLKSHRFPCHWSWVANTLNPYHEANDDLMMIMDDRLIANTLHWWYFGPDNTGDI MTLKFLTGLQNFISSMATVHLVTADGSFDCQGNPGEQEALVSSLHYCEVVTALTTLGNGGSFVLKMFTMF EHCSINLMYLLNCCFDQVHVFKPATSKAGNSEVYVVCLHYKGREAIHPLLSKMTLNFGTEMKRKALFPHH VIPDSFLKRHEECCVFFHKYQLETISENIRLFECMGKAEQEKLNNLRDCAIQYFMQKFQLKHLSRNNWLV 40 KKSSIGCSTNTKWFGQRNKYFKTYNERKMLEALSWKDKVAKGYFNSWAEEHGVYHPGQSSILEGTASNLE 8 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CHLWHILEGKKLPKVKCSPFCNGEILKTLNEAIEKSLGGAFNLDSKFRPKQQYSCSCHVFSEELIFSELC SLTECLQDEQVVVPSNQIKCLLVGFSTLRNIKMHIPLEVRLLESAELTTFSCSLLHDGDPTYQRLFLDCL LHSLRELHTGDVMILPVLSCFTRFMAGLIFVLHSCFRFITFVCPTSSDPLRTCAVLLCVGYQDLPNPVFR YLQSVNELLSTLLNSDSPQQVLQFVPMEVLLKGALLDFLWDLNAAIAKRHLHFIIQREREEIINSLQLQN 5 >NP_001311307.1 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2 [Homo sapiens] MSKCRKTPVQQLASPASFSPDILADIFELFAKNFSYGKPLNNEWQLPDPSEIFTCDHTELNAFLDLKNSL NEVKNLLSDKKLDEWHEHTAFTNKAGKIISHVRKSVNAELCTQAWCKFHEILCSFPLIPQEAFQNGKLNS 10 LHLCEAPGAFIASLNHYLKSHRFPCHWSWVANTLNPYHEANDDLMMIMDDRLIANTLHWWYFGPDNTGDI MTLKFLTGLQNFISSMATVHLVTADGSFDCQGNPGEQEALVSSLHYCEVVTALTTLGNGGSFVLKMFTMF EHCSINLMYLLNCCFDQVHVFKPATSKAGNSEVYVVCLHYKGREAIHPLLSKMTLNFGTEMKRKALFPHH VIPDSFLKRHEECCVFFHKYQLETISENIRLFECMGKAEQEKLNNLRDCAIQYFMQKFQLKHLSRNNWLV KKSSIGCSTNTKWFGQRNKYFKTYNERKMLEALSWKDKVAKGYFNSWAEEHGVYHPGQSSILEGTASNLE 15 CHLWHILEGKKLPKVKCSPFCNGEILKTLNEAIEKSLGGAFNLDSKFRPKQQYSCSCHVFSEELIFSELC SLTECLQDEQVVVPSNQIKCLLVGFSTLRNIKMHIPLEVRLLESAELTTFSCSLLHDGDPTYQRLFLDCL LHSLRELHTGDVMILPVLSCFTRFMAGLIFVLHSCFRFITFVCPTSSDPLRTCAVLLCVGYQDLPNPVFR YLQSVNELLSTLLNSDSPQQVLQFVPMEVLLKGALLDFLWDLNAAIAKRHLHFIIQREREEIINSLQLQN 20 >NP_001311308.1 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2 [Homo sapiens] MSKCRKTPVQQLASPASFSPDILADIFELFAKNFSYGKPLNNEWQLPDPSEIFTCDHTELNAFLDLKNSL NEVKNLLSDKKLDEWHEHTAFTNKAGKIISHVRKSVNAELCTQAWCKFHEILCSFPLIPQEAFQNGKLNS LHLCEAPGAFIASLNHYLKSHRFPCHWSWVANTLNPYHEANDDLMMIMDDRLIANTLHWWYFGPDNTGDI 25 MTLKFLTGLQNFISSMATVHLVTADGSFDCQGNPGEQEALVSSLHYCEVVTALTTLGNGGSFVLKMFTMF EHCSINLMYLLNCCFDQVHVFKPATSKAGNSEVYVVCLHYKGREAIHPLLSKMTLNFGTEMKRKALFPHH VIPDSFLKRHEECCVFFHKYQLETISENIRLFECMGKAEQEKLNNLRDCAIQYFMQKFQLKHLSRNNWLV KKSSIGCSTNTKWFGQRNKYFKTYNERKMLEALSWKDKVAKGYFNSWAEEHGVYHPGQSSILEGTASNLE CHLWHILEGKKLPKVKCSPFCNGEILKTLNEAIEKSLGGAFNLDSKFRPKQQYSCSCHVFSEELIFSELC 30 SLTECLQDEQVVVPSNQIKCLLVGFSTLRNIKMHIPLEVRLLESAELTTFSCSLLHDGDPTYQRLFLDCL LHSLRELHTGDVMILPVLSCFTRFMAGLIFVLHSCFRFITFVCPTSSDPLRTCAVLLCVGYQDLPNPVFR YLQSVNELLSTLLNSDSPQQVLQFVPMEVLLKGALLDFLWDLNAAIAKRHLHFIIQREREEIINSLQLQN >NP_060818.4 cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2 [Homo 35 sapiens] MSKCRKTPVQQLASPASFSPDILADIFELFAKNFSYGKPLNNEWQLPDPSEIFTCDHTELNAFLDLKNSL NEVKNLLSDKKLDEWHEHTAFTNKAGKIISHVRKSVNAELCTQAWCKFHEILCSFPLIPQEAFQNGKLNS LHLCEAPGAFIASLNHYLKSHRFPCHWSWVANTLNPYHEANDDLMMIMDDRLIANTLHWWYFGPDNTGDI MTLKFLTGLQNFISSMATVHLVTADGSFDCQGNPGEQEALVSSLHYCEVVTALTTLGNGGSFVLKMFTMF 40 EHCSINLMYLLNCCFDQVHVFKPATSKAGNSEVYVVCLHYKGREAIHPLLSKMTLNFGTEMKRKALFPHH VIPDSFLKRHEECCVFFHKYQLETISENIRLFECMGKAEQEKLNNLRDCAIQYFMQKFQLKHLSRNNWLV 9 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 KKSSIGCSTNTKWFGQRNKYFKTYNERKMLEALSWKDKVAKGYFNSWAEEHGVYHPGQSSILEGTASNLE CHLWHILEGKKLPKVKCSPFCNGEILKTLNEAIEKSLGGAFNLDSKFRPKQQYSCSCHVFSEELIFSELC SLTECLQDEQVVVPSNQIKCLLVGFSTLRNIKMHIPLEVRLLESAELTTFSCSLLHDGDPTYQRLFLDCL LHSLRELHTGDVMILPVLSCFTRFMAGLIFVLHSCFRFITFVCPTSSDPLRTCAVLLCVGYQDLPNPVFR 5 YLQSVNELLSTLLNSDSPQQVLQFVPMEVLLKGALLDFLWDLNAAIAKRHLHFIIQREREEIINSLQLQN By “cap methyltransferase 2 polynucleotide” or “CMTR2 polynucleotide” is meant a nucleic acid molecule encoding an CMTR2 polypeptide or fragment thereof. The sequences of exemplary CMTR2 polynucleotides are provided below: 10 >NM_001099642.2 Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 2, mRNA GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGGTGGGACTGGCTTCCCGGT GCCGCGAGGGCGGGTCCGGACAGCCTTCCCCCCAGTCCGGCGCACCATCTCCCTGCCTTGTGGCTGGAGG CGCCGCGGACCCAAAGGGAGGGACCATCCCGGGAAGCAGCCCCGAGAGCGGAAGTGCAGAATGGCTTCCT 15 CGAGAGAGTAAAGTGCAGCCTCTCCAGACACTGGGGCCCCAGTGGGCGTGGGCGAAGGTAATCCAGGCCT GGGTACGATTCCGGGCCCTCCTTCGACTTCCCAGCGGTTGCTGGTAGGAGGAGTTGGCGGAAGCACTTGG AACTCCTTTATAAGTGTCAGCTGTGAGATTTTAATTTGATTTGAAAATGAGTAAGTGCAGAAAGACACCA GTTCAGCAGCTAGCAAGTCCCGCGTCATTCAGCCCAGATATTCTTGCTGACATTTTTGAACTCTTTGCCA AGAACTTTTCTTATGGCAAGCCACTTAATAATGAGTGGCAGTTACCAGATCCCAGTGAGATTTTCACCTG 20 TGACCACACTGAACTTAATGCATTTCTTGATTTGAAGAACTCCCTAAATGAAGTAAAAAACCTACTGAGT GATAAGAAACTGGATGAGTGGCATGAGCACACTGCTTTCACTAATAAAGCGGGGAAAATCATTTCTCATG TTAGAAAATCTGTGAATGCTGAACTTTGTACTCAAGCATGGTGTAAGTTCCATGAGATTTTGTGCAGCTT TCCACTTATTCCACAGGAAGCTTTTCAGAATGGAAAACTGAATTCTCTACACCTTTGTGAAGCTCCAGGA GCTTTTATAGCTAGTCTCAACCACTACTTAAAATCCCATCGGTTTCCTTGTCATTGGAGTTGGGTAGCGA 25 ATACTCTGAATCCATACCATGAAGCAAATGACGACCTCATGATGATTATGGATGACCGGCTTATTGCAAA TACCTTGCACTGGTGGTACTTTGGTCCAGATAACACTGGTGATATCATGACCCTGAAATTCTTGACTGGA CTTCAGAATTTCATAAGCAGCATGGCTACTGTTCACTTGGTCACTGCAGATGGGAGTTTTGATTGCCAAG GAAACCCAGGTGAACAAGAAGCTTTAGTTTCTTCTTTGCATTACTGTGAAGTTGTCACTGCTCTGACCAC TCTTGGAAACGGTGGCTCTTTTGTTCTAAAGATGTTTACTATGTTTGAACATTGTTCCATAAACTTGATG 30 TACCTGCTAAACTGTTGTTTTGACCAAGTCCATGTTTTCAAACCTGCTACTAGCAAGGCAGGAAACTCCG AAGTCTATGTGGTTTGCCTCCACTATAAGGGGAGAGAGGCCATCCATCCTCTGTTATCTAAGATGACCTT GAATTTTGGGACTGAAATGAAAAGGAAAGCCCTTTTTCCCCATCATGTGATTCCTGATTCTTTTCTTAAG AGACATGAAGAATGTTGTGTGTTCTTTCATAAATATCAGCTAGAGACTATTTCTGAAAACATTCGTCTAT TTGAGTGCATGGGAAAGGCGGAACAAGAAAAGCTGAATAATTTAAGGGATTGTGCTATACAATATTTTAT 35 GCAAAAATTTCAACTGAAACATCTTTCCAGAAATAATTGGCTAGTAAAAAAATCTAGTATTGGTTGTAGT ACAAATACAAAATGGTTTGGGCAGAGGAACAAATATTTTAAAACTTATAATGAAAGGAAGATGCTAGAAG CCCTTTCATGGAAAGATAAAGTAGCCAAAGGATACTTTAATAGTTGGGCTGAAGAACATGGTGTATATCA TCCTGGGCAGAGTTCTATTTTAGAAGGAACAGCTTCCAATCTTGAGTGTCACTTATGGCATATTTTGGAG GGAAAGAAACTGCCAAAGGTAAAATGTTCTCCTTTTTGCAATGGTGAAATTTTAAAAACTCTTAATGAAG 40 CAATTGAAAAGTCATTAGGAGGAGCTTTTAATTTGGATTCCAAGTTTAGGCCAAAACAGCAGTATTCTTG 10 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TTCTTGTCATGTTTTTTCTGAAGAACTGATATTTTCCGAGTTGTGTAGCCTTACTGAGTGCCTTCAGGAT GAGCAGGTTGTAGTACCCAGCAATCAAATAAAGTGCCTGCTGGTGGGCTTTTCGACTCTCCGTAATATCA AAATGCATATACCGTTGGAAGTTCGACTCCTAGAATCAGCTGAACTCACAACTTTTAGCTGTTCATTGCT TCATGATGGAGATCCAACTTACCAGCGTTTATTTTTGGACTGCCTTCTACATTCATTGCGGGAGCTTCAT 5 ACAGGAGATGTTATGATTTTGCCTGTACTTTCTTGCTTCACAAGATTTATGGCTGGTTTGATCTTTGTAC TCCACAGTTGTTTTAGATTCATCACTTTTGTTTGTCCCACATCCTCTGATCCCCTGAGGACCTGCGCAGT CCTGCTATGTGTTGGTTATCAGGACCTTCCAAATCCAGTTTTCCGATATTTGCAGAGTGTGAATGAATTG TTGAGCACTTTGCTCAACTCTGACTCACCCCAGCAGGTTTTACAGTTTGTGCCAATGGAGGTACTCCTTA AGGGGGCCCTGCTTGATTTTTTGTGGGATTTGAATGCTGCCATTGCTAAAAGGCATTTGCATTTCATTAT 10 TCAAAGAGAGAGAGAAGAAATTATCAACAGCCTTCAGTTACAAAACTGAACATATGCTTTCTGAGATTCA ACTTTATGATTTCTTATAATTTGCCCAGTATTTGCATCCTGTTGCTCTATTAATTTAAAAACCTTTTATT TTGGGGAAAGGCCAACATTTGCATCATTCAAAGTCTCATTAATTCTGGAAAACCATCCATTCTGATCTCT AGGGTATATACACCCACAGGCATAGAGCTCTTCCACGTGGTGGAATCTATGCAATGATAGATATTCACAC TCTAAATATGAGGTGTGTGTATGTGTATGGGTGGCCACAGCCATGCTTACCTATGCCATTTAGTTGGTCT 15 TACTTAATCTGCTTAAGATTTGCATCTGTGTACCTTTGTTCAGATTAGTTTTTTTTTTCCAGCCGATTTC CTCTTAGTGGCTAATGCTGTTAGTGAATTTTCCAACTAATTTCCTCTCATTGGTTAATGTTGTTAATGAA TTGAGAGAGGTAATTGAGGAAAGGAAATGAGTAAATCACTGTTCAGCAACACTGATTTCCGTTAACACAT CAGTTATGAATTTCAGGGAATTCATCTCGCCAGATTCTTGATAACATGCCATTCATTGCCCTTAGGTGAT TGACCCTATTTTCTTACATGGCTCAAATAAAACTAGTATGCTGTTGTATGAATCTTTTACTGACCACACC 20 ATCCAACTATAAAAATATAACGGGACAGCTTTAAACCAAAGATCATGTTTAGAACAATGAAAAATTATTT GTTGTATCTAATACACGCCTGTATTGTGAAAAGCTTCATTTAGCAATGATGTAATAATTTTTAACTTCCA GGAAATAATCTGTGAATGGAAAGATTTTTTAAGATTTTGAGATAGTGTTTAGTCTCATGTTGGGAACACA TGAATGTGATGAACATAGTGAATACTAAAGAAAACGCTTCAGACTTTCAGAATGATGGTTCAGAATTTAA AATTTTTAATCTTTTCTAATTTCTTTTTTTCAGTGTGAAAATAGCACTTTACCAAAAGATTAGCCATGAA 25 ATGGTTATTTTGCCAGTTACATTTGATTTCTTTTGTATCTGCAATGTAATGAGTTATTTTATTTCTTCTG TATTTGCAGTGTAATGAGTTTTTGTGGCAAAGTGTATTAAGCAATTTTTCATTATCTTGAAGTTCCACAA AGTGGAGAATATTTATATTCTCACATGCATTTTAGGCACTTTTGATATGTGAAAATAGATGTATTTTCTG ATGCATTTGGTTAATAAATATTAATCTGAACATTTTCATGTTCTTTGCTATTTTGAATTCCATTATAGAT TCATGAATAAAGTCATTACTAGAGAGATTTTGTGTTCATCTTTTTTAAATGGAATTATGGGAGAAGTTAA 30 AAATATAAGTTGGAAACAGACATTTTTAAAGGGAGTTTTGAATGATAGTTAATGTTCTTTCATTTTCTCT GATTTGCATTCATTAAAATTTAGTCCTTAATATTGTAATACTTATGGAAGGCCTACTTCAGGAAAGAAAT TGAATACTTAAGGGGGATTGGAAGAGGAGTGTGTGGAGGCCTGGAAGTAGATTTTTGAAAGAAATCTGAT TTCCTACTGGTGACTCTATGGAAGCAATTCCAGTTGTATGACGATGGAAGTCTGACAGAAGGTTGATTAC CAGGAGGAAAAAAAAAAACACTCTTGGAAAACCATTTTGCACTTAATAGTCTTTATGCCTTGCTAAGTGG 35 GATTCAATATACTAATAATGCAGCTTATTTTTTACTTATTTTAACTATTTGATAAGAATGAAGTTGAAGG CATGGGTCCAGATTCTAGCATTATTACTGTCTTTAAGCCCTTCAAATAAGTATTGAATGCTTAGTGTTTT GTTGACACTAATCTGTACTGGAATATATAACACTGAAGTATTCTCAAATGTATTTTGCTTTTTAACGTGG GTTTTGGAATCAATTGCTCATCTGATTCATTGGATTCAAATTGCTTTAGAGGTTAGACATGACTTGAGGA AGGTTCAGATTGAACTGGATATATTAATAGATGACAGGTACTAATGATTTATATGAGTTTCTCTTTAAGA 40 GAAAGGAGAGGAAAAATGCTATTCAAAAGATGAATGGAAAAGTAGAAGAAGACAGGAAGAGGTGAGAGTA TGCATGTTTTACCAAGGTAGACTGTTCTTAAAAACTTTTTTCCACAGGTTTTGAGATTTTTAATTTGTAA 11 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TATCTATTAAAAAACAAACATCAAA >NM_001324374.2 Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 3, mRNA 5 GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGTTGCTGGTAGGAGGAGTTG GCGGAAGCACTTGGAACTCCTTTATAAGTGTCAGCTGTGAGATTTTAATTTGATTTGAAAATGAGTAAGT GCAGAAAGACACCAGTTCAGCAGCTAGCAAGTCCCGCGTCATTCAGCCCAGATATTCTTGCTGACATTTT TGAACTCTTTGCCAAGAACTTTTCTTATGGCAAGCCACTTAATAATGAGTGGCAGTTACCAGATCCCAGT GAGATTTTCACCTGTGACCACACTGAACTTAATGCATTTCTTGATTTGAAGAACTCCCTAAATGAAGTAA 10 AAAACCTACTGAGTGATAAGAAACTGGATGAGTGGCATGAGCACACTGCTTTCACTAATAAAGCGGGGAA AATCATTTCTCATGTTAGAAAATCTGTGAATGCTGAACTTTGTACTCAAGCATGGTGTAAGTTCCATGAG ATTTTGTGCAGCTTTCCACTTATTCCACAGGAAGCTTTTCAGAATGGAAAACTGAATTCTCTACACCTTT GTGAAGCTCCAGGAGCTTTTATAGCTAGTCTCAACCACTACTTAAAATCCCATCGGTTTCCTTGTCATTG GAGTTGGGTAGCGAATACTCTGAATCCATACCATGAAGCAAATGACGACCTCATGATGATTATGGATGAC 15 CGGCTTATTGCAAATACCTTGCACTGGTGGTACTTTGGTCCAGATAACACTGGTGATATCATGACCCTGA AATTCTTGACTGGACTTCAGAATTTCATAAGCAGCATGGCTACTGTTCACTTGGTCACTGCAGATGGGAG TTTTGATTGCCAAGGAAACCCAGGTGAACAAGAAGCTTTAGTTTCTTCTTTGCATTACTGTGAAGTTGTC ACTGCTCTGACCACTCTTGGAAACGGTGGCTCTTTTGTTCTAAAGATGTTTACTATGTTTGAACATTGTT CCATAAACTTGATGTACCTGCTAAACTGTTGTTTTGACCAAGTCCATGTTTTCAAACCTGCTACTAGCAA 20 GGCAGGAAACTCCGAAGTCTATGTGGTTTGCCTCCACTATAAGGGGAGAGAGGCCATCCATCCTCTGTTA TCTAAGATGACCTTGAATTTTGGGACTGAAATGAAAAGGAAAGCCCTTTTTCCCCATCATGTGATTCCTG ATTCTTTTCTTAAGAGACATGAAGAATGTTGTGTGTTCTTTCATAAATATCAGCTAGAGACTATTTCTGA AAACATTCGTCTATTTGAGTGCATGGGAAAGGCGGAACAAGAAAAGCTGAATAATTTAAGGGATTGTGCT ATACAATATTTTATGCAAAAATTTCAACTGAAACATCTTTCCAGAAATAATTGGCTAGTAAAAAAATCTA 25 GTATTGGTTGTAGTACAAATACAAAATGGTTTGGGCAGAGGAACAAATATTTTAAAACTTATAATGAAAG GAAGATGCTAGAAGCCCTTTCATGGAAAGATAAAGTAGCCAAAGGATACTTTAATAGTTGGGCTGAAGAA CATGGTGTATATCATCCTGGGCAGAGTTCTATTTTAGAAGGAACAGCTTCCAATCTTGAGTGTCACTTAT GGCATATTTTGGAGGGAAAGAAACTGCCAAAGGTAAAATGTTCTCCTTTTTGCAATGGTGAAATTTTAAA AACTCTTAATGAAGCAATTGAAAAGTCATTAGGAGGAGCTTTTAATTTGGATTCCAAGTTTAGGCCAAAA 30 CAGCAGTATTCTTGTTCTTGTCATGTTTTTTCTGAAGAACTGATATTTTCCGAGTTGTGTAGCCTTACTG AGTGCCTTCAGGATGAGCAGGTTGTAGTACCCAGCAATCAAATAAAGTGCCTGCTGGTGGGCTTTTCGAC TCTCCGTAATATCAAAATGCATATACCGTTGGAAGTTCGACTCCTAGAATCAGCTGAACTCACAACTTTT AGCTGTTCATTGCTTCATGATGGAGATCCAACTTACCAGCGTTTATTTTTGGACTGCCTTCTACATTCAT TGCGGGAGCTTCATACAGGAGATGTTATGATTTTGCCTGTACTTTCTTGCTTCACAAGATTTATGGCTGG 35 TTTGATCTTTGTACTCCACAGTTGTTTTAGATTCATCACTTTTGTTTGTCCCACATCCTCTGATCCCCTG AGGACCTGCGCAGTCCTGCTATGTGTTGGTTATCAGGACCTTCCAAATCCAGTTTTCCGATATTTGCAGA GTGTGAATGAATTGTTGAGCACTTTGCTCAACTCTGACTCACCCCAGCAGGTTTTACAGTTTGTGCCAAT GGAGGTACTCCTTAAGGGGGCCCTGCTTGATTTTTTGTGGGATTTGAATGCTGCCATTGCTAAAAGGCAT TTGCATTTCATTATTCAAAGAGAGAGAGAAGAAATTATCAACAGCCTTCAGTTACAAAACTGAACATATG 40 CTTTCTGAGATTCAACTTTATGATTTCTTATAATTTGCCCAGTATTTGCATCCTGTTGCTCTATTAATTT AAAAACCTTTTATTTTGGGGAAAGGCCAACATTTGCATCATTCAAAGTCTCATTAATTCTGGAAAACCAT 12 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CCATTCTGATCTCTAGGGTATATACACCCACAGGCATAGAGCTCTTCCACGTGGTGGAATCTATGCAATG ATAGATATTCACACTCTAAATATGAGGTGTGTGTATGTGTATGGGTGGCCACAGCCATGCTTACCTATGC CATTTAGTTGGTCTTACTTAATCTGCTTAAGATTTGCATCTGTGTACCTTTGTTCAGATTAGTTTTTTTT TTCCAGCCGATTTCCTCTTAGTGGCTAATGCTGTTAGTGAATTTTCCAACTAATTTCCTCTCATTGGTTA 5 ATGTTGTTAATGAATTGAGAGAGGTAATTGAGGAAAGGAAATGAGTAAATCACTGTTCAGCAACACTGAT TTCCGTTAACACATCAGTTATGAATTTCAGGGAATTCATCTCGCCAGATTCTTGATAACATGCCATTCAT TGCCCTTAGGTGATTGACCCTATTTTCTTACATGGCTCAAATAAAACTAGTATGCTGTTGTATGAATCTT TTACTGACCACACCATCCAACTATAAAAATATAACGGGACAGCTTTAAACCAAAGATCATGTTTAGAACA ATGAAAAATTATTTGTTGTATCTAATACACGCCTGTATTGTGAAAAGCTTCATTTAGCAATGATGTAATA 10 ATTTTTAACTTCCAGGAAATAATCTGTGAATGGAAAGATTTTTTAAGATTTTGAGATAGTGTTTAGTCTC ATGTTGGGAACACATGAATGTGATGAACATAGTGAATACTAAAGAAAACGCTTCAGACTTTCAGAATGAT GGTTCAGAATTTAAAATTTTTAATCTTTTCTAATTTCTTTTTTTCAGTGTGAAAATAGCACTTTACCAAA AGATTAGCCATGAAATGGTTATTTTGCCAGTTACATTTGATTTCTTTTGTATCTGCAATGTAATGAGTTA TTTTATTTCTTCTGTATTTGCAGTGTAATGAGTTTTTGTGGCAAAGTGTATTAAGCAATTTTTCATTATC 15 TTGAAGTTCCACAAAGTGGAGAATATTTATATTCTCACATGCATTTTAGGCACTTTTGATATGTGAAAAT AGATGTATTTTCTGATGCATTTGGTTAATAAATATTAATCTGAACATTTTCATGTTCTTTGCTATTTTGA ATTCCATTATAGATTCATGAATAAAGTCATTACTAGAGAGATTTTGTGTTCATCTTTTTTAAATGGAATT ATGGGAGAAGTTAAAAATATAAGTTGGAAACAGACATTTTTAAAGGGAGTTTTGAATGATAGTTAATGTT CTTTCATTTTCTCTGATTTGCATTCATTAAAATTTAGTCCTTAATATTGTAATACTTATGGAAGGCCTAC 20 TTCAGGAAAGAAATTGAATACTTAAGGGGGATTGGAAGAGGAGTGTGTGGAGGCCTGGAAGTAGATTTTT GAAAGAAATCTGATTTCCTACTGGTGACTCTATGGAAGCAATTCCAGTTGTATGACGATGGAAGTCTGAC AGAAGGTTGATTACCAGGAGGAAAAAAAAAAACACTCTTGGAAAACCATTTTGCACTTAATAGTCTTTAT GCCTTGCTAAGTGGGATTCAATATACTAATAATGCAGCTTATTTTTTACTTATTTTAACTATTTGATAAG AATGAAGTTGAAGGCATGGGTCCAGATTCTAGCATTATTACTGTCTTTAAGCCCTTCAAATAAGTATTGA 25 ATGCTTAGTGTTTTGTTGACACTAATCTGTACTGGAATATATAACACTGAAGTATTCTCAAATGTATTTT GCTTTTTAACGTGGGTTTTGGAATCAATTGCTCATCTGATTCATTGGATTCAAATTGCTTTAGAGGTTAG ACATGACTTGAGGAAGGTTCAGATTGAACTGGATATATTAATAGATGACAGGTACTAATGATTTATATGA GTTTCTCTTTAAGAGAAAGGAGAGGAAAAATGCTATTCAAAAGATGAATGGAAAAGTAGAAGAAGACAGG AAGAGGTGAGAGTATGCATGTTTTACCAAGGTAGACTGTTCTTAAAAACTTTTTTCCACAGGTTTTGAGA 30 TTTTTAATTTGTAATATCTATTAAAAAACAAACATCAAA >NM_001324377.2 Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 4, mRNA AGCCAGTGAGCGGAACCAGCACGTCCTGACAGACAGTTGCTGGTAGGAGGAGTTGGCGGAAGCACTTGGA 35 ACTCCTTTATAAGTGTCAGCTGTGAGATTTTAATTTGATTTGAAAATGAGTAAGTGCAGAAAGACACCAG TTCAGCAGCTAGCAAGTCCCGCGTCATTCAGCCCAGATATTCTTGCTGACATTTTTGAACTCTTTGCCAA GAACTTTTCTTATGGCAAGCCACTTAATAATGAGTGGCAGTTACCAGATCCCAGTGAGATTTTCACCTGT GACCACACTGAACTTAATGCATTTCTTGATTTGAAGAACTCCCTAAATGAAGTAAAAAACCTACTGAGTG ATAAGAAACTGGATGAGTGGCATGAGCACACTGCTTTCACTAATAAAGCGGGGAAAATCATTTCTCATGT 40 TAGAAAATCTGTGAATGCTGAACTTTGTACTCAAGCATGGTGTAAGTTCCATGAGATTTTGTGCAGCTTT CCACTTATTCCACAGGAAGCTTTTCAGAATGGAAAACTGAATTCTCTACACCTTTGTGAAGCTCCAGGAG 13 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CTTTTATAGCTAGTCTCAACCACTACTTAAAATCCCATCGGTTTCCTTGTCATTGGAGTTGGGTAGCGAA TACTCTGAATCCATACCATGAAGCAAATGACGACCTCATGATGATTATGGATGACCGGCTTATTGCAAAT ACCTTGCACTGGTGGTACTTTGGTCCAGATAACACTGGTGATATCATGACCCTGAAATTCTTGACTGGAC TTCAGAATTTCATAAGCAGCATGGCTACTGTTCACTTGGTCACTGCAGATGGGAGTTTTGATTGCCAAGG 5 AAACCCAGGTGAACAAGAAGCTTTAGTTTCTTCTTTGCATTACTGTGAAGTTGTCACTGCTCTGACCACT CTTGGAAACGGTGGCTCTTTTGTTCTAAAGATGTTTACTATGTTTGAACATTGTTCCATAAACTTGATGT ACCTGCTAAACTGTTGTTTTGACCAAGTCCATGTTTTCAAACCTGCTACTAGCAAGGCAGGAAACTCCGA AGTCTATGTGGTTTGCCTCCACTATAAGGGGAGAGAGGCCATCCATCCTCTGTTATCTAAGATGACCTTG AATTTTGGGACTGAAATGAAAAGGAAAGCCCTTTTTCCCCATCATGTGATTCCTGATTCTTTTCTTAAGA 10 GACATGAAGAATGTTGTGTGTTCTTTCATAAATATCAGCTAGAGACTATTTCTGAAAACATTCGTCTATT TGAGTGCATGGGAAAGGCGGAACAAGAAAAGCTGAATAATTTAAGGGATTGTGCTATACAATATTTTATG CAAAAATTTCAACTGAAACATCTTTCCAGAAATAATTGGCTAGTAAAAAAATCTAGTATTGGTTGTAGTA CAAATACAAAATGGTTTGGGCAGAGGAACAAATATTTTAAAACTTATAATGAAAGGAAGATGCTAGAAGC CCTTTCATGGAAAGATAAAGTAGCCAAAGGATACTTTAATAGTTGGGCTGAAGAACATGGTGTATATCAT 15 CCTGGGCAGAGTTCTATTTTAGAAGGAACAGCTTCCAATCTTGAGTGTCACTTATGGCATATTTTGGAGG GAAAGAAACTGCCAAAGGTAAAATGTTCTCCTTTTTGCAATGGTGAAATTTTAAAAACTCTTAATGAAGC AATTGAAAAGTCATTAGGAGGAGCTTTTAATTTGGATTCCAAGTTTAGGCCAAAACAGCAGTATTCTTGT TCTTGTCATGTTTTTTCTGAAGAACTGATATTTTCCGAGTTGTGTAGCCTTACTGAGTGCCTTCAGGATG AGCAGGTTGTAGTACCCAGCAATCAAATAAAGTGCCTGCTGGTGGGCTTTTCGACTCTCCGTAATATCAA 20 AATGCATATACCGTTGGAAGTTCGACTCCTAGAATCAGCTGAACTCACAACTTTTAGCTGTTCATTGCTT CATGATGGAGATCCAACTTACCAGCGTTTATTTTTGGACTGCCTTCTACATTCATTGCGGGAGCTTCATA CAGGAGATGTTATGATTTTGCCTGTACTTTCTTGCTTCACAAGATTTATGGCTGGTTTGATCTTTGTACT CCACAGTTGTTTTAGATTCATCACTTTTGTTTGTCCCACATCCTCTGATCCCCTGAGGACCTGCGCAGTC CTGCTATGTGTTGGTTATCAGGACCTTCCAAATCCAGTTTTCCGATATTTGCAGAGTGTGAATGAATTGT 25 TGAGCACTTTGCTCAACTCTGACTCACCCCAGCAGGTTTTACAGTTTGTGCCAATGGAGGTACTCCTTAA GGGGGCCCTGCTTGATTTTTTGTGGGATTTGAATGCTGCCATTGCTAAAAGGCATTTGCATTTCATTATT CAAAGAGAGAGAGAAGAAATTATCAACAGCCTTCAGTTACAAAACTGAACATATGCTTTCTGAGATTCAA CTTTATGATTTCTTATAATTTGCCCAGTATTTGCATCCTGTTGCTCTATTAATTTAAAAACCTTTTATTT TGGGGAAAGGCCAACATTTGCATCATTCAAAGTCTCATTAATTCTGGAAAACCATCCATTCTGATCTCTA 30 GGGTATATACACCCACAGGCATAGAGCTCTTCCACGTGGTGGAATCTATGCAATGATAGATATTCACACT CTAAATATGAGGTGTGTGTATGTGTATGGGTGGCCACAGCCATGCTTACCTATGCCATTTAGTTGGTCTT ACTTAATCTGCTTAAGATTTGCATCTGTGTACCTTTGTTCAGATTAGTTTTTTTTTTCCAGCCGATTTCC TCTTAGTGGCTAATGCTGTTAGTGAATTTTCCAACTAATTTCCTCTCATTGGTTAATGTTGTTAATGAAT TGAGAGAGGTAATTGAGGAAAGGAAATGAGTAAATCACTGTTCAGCAACACTGATTTCCGTTAACACATC 35 AGTTATGAATTTCAGGGAATTCATCTCGCCAGATTCTTGATAACATGCCATTCATTGCCCTTAGGTGATT GACCCTATTTTCTTACATGGCTCAAATAAAACTAGTATGCTGTTGTATGAATCTTTTACTGACCACACCA TCCAACTATAAAAATATAACGGGACAGCTTTAAACCAAAGATCATGTTTAGAACAATGAAAAATTATTTG TTGTATCTAATACACGCCTGTATTGTGAAAAGCTTCATTTAGCAATGATGTAATAATTTTTAACTTCCAG GAAATAATCTGTGAATGGAAAGATTTTTTAAGATTTTGAGATAGTGTTTAGTCTCATGTTGGGAACACAT 40 GAATGTGATGAACATAGTGAATACTAAAGAAAACGCTTCAGACTTTCAGAATGATGGTTCAGAATTTAAA ATTTTTAATCTTTTCTAATTTCTTTTTTTCAGTGTGAAAATAGCACTTTACCAAAAGATTAGCCATGAAA 14 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGGTTATTTTGCCAGTTACATTTGATTTCTTTTGTATCTGCAATGTAATGAGTTATTTTATTTCTTCTGT ATTTGCAGTGTAATGAGTTTTTGTGGCAAAGTGTATTAAGCAATTTTTCATTATCTTGAAGTTCCACAAA GTGGAGAATATTTATATTCTCACATGCATTTTAGGCACTTTTGATATGTGAAAATAGATGTATTTTCTGA TGCATTTGGTTAATAAATATTAATCTGAACATTTTCATGTTCTTTGCTATTTTGAATTCCATTATAGATT 5 CATGAATAAAGTCATTACTAGAGAGATTTTGTGTTCATCTTTTTTAAATGGAATTATGGGAGAAGTTAAA AATATAAGTTGGAAACAGACATTTTTAAAGGGAGTTTTGAATGATAGTTAATGTTCTTTCATTTTCTCTG ATTTGCATTCATTAAAATTTAGTCCTTAATATTGTAATACTTATGGAAGGCCTACTTCAGGAAAGAAATT GAATACTTAAGGGGGATTGGAAGAGGAGTGTGTGGAGGCCTGGAAGTAGATTTTTGAAAGAAATCTGATT TCCTACTGGTGACTCTATGGAAGCAATTCCAGTTGTATGACGATGGAAGTCTGACAGAAGGTTGATTACC 10 AGGAGGAAAAAAAAAAACACTCTTGGAAAACCATTTTGCACTTAATAGTCTTTATGCCTTGCTAAGTGGG ATTCAATATACTAATAATGCAGCTTATTTTTTACTTATTTTAACTATTTGATAAGAATGAAGTTGAAGGC ATGGGTCCAGATTCTAGCATTATTACTGTCTTTAAGCCCTTCAAATAAGTATTGAATGCTTAGTGTTTTG TTGACACTAATCTGTACTGGAATATATAACACTGAAGTATTCTCAAATGTATTTTGCTTTTTAACGTGGG TTTTGGAATCAATTGCTCATCTGATTCATTGGATTCAAATTGCTTTAGAGGTTAGACATGACTTGAGGAA 15 GGTTCAGATTGAACTGGATATATTAATAGATGACAGGTACTAATGATTTATATGAGTTTCTCTTTAAGAG AAAGGAGAGGAAAAATGCTATTCAAAAGATGAATGGAAAAGTAGAAGAAGACAGGAAGAGGTGAGAGTAT GCATGTTTTACCAAGGTAGACTGTTCTTAAAAACTTTTTTCCACAGGTTTTGAGATTTTTAATTTGTAAT ATCTATTAAAAAACAAACATCAAA 20 >NM_001324378.2 Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 5, mRNA GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGGTGGGACTGGCTTCCCGGT GCCGCGAGGGCGGGTCCGGACAGCCTTCCCCCCAGTCCGGCGCACCATCTCCCTGCCTTGTGGCTGGAGG CGCCGCGGACCCAAAGGGAGGGACCATCCCGGGAAGCAGCCCCGAGAGCGGAAGTGCAGAATGGCTTCCT 25 CGAGAGAGTAAAGTGCAGCCTCTCCAGACACTGGGGCCCCAGTGGGCGTGGGCGAAGGTAATCCAGGCCT GGGTACGATTCCGGGCCCTCCTTCGACTTCCCAGCGGATTTTAATTTGATTTGAAAATGAGTAAGTGCAG AAAGACACCAGTTCAGCAGCTAGCAAGTCCCGCGTCATTCAGCCCAGATATTCTTGCTGACATTTTTGAA CTCTTTGCCAAGAACTTTTCTTATGGCAAGCCACTTAATAATGAGTGGCAGTTACCAGATCCCAGTGAGA TTTTCACCTGTGACCACACTGAACTTAATGCATTTCTTGATTTGAAGAACTCCCTAAATGAAGTAAAAAA 30 CCTACTGAGTGATAAGAAACTGGATGAGTGGCATGAGCACACTGCTTTCACTAATAAAGCGGGGAAAATC ATTTCTCATGTTAGAAAATCTGTGAATGCTGAACTTTGTACTCAAGCATGGTGTAAGTTCCATGAGATTT TGTGCAGCTTTCCACTTATTCCACAGGAAGCTTTTCAGAATGGAAAACTGAATTCTCTACACCTTTGTGA AGCTCCAGGAGCTTTTATAGCTAGTCTCAACCACTACTTAAAATCCCATCGGTTTCCTTGTCATTGGAGT TGGGTAGCGAATACTCTGAATCCATACCATGAAGCAAATGACGACCTCATGATGATTATGGATGACCGGC 35 TTATTGCAAATACCTTGCACTGGTGGTACTTTGGTCCAGATAACACTGGTGATATCATGACCCTGAAATT CTTGACTGGACTTCAGAATTTCATAAGCAGCATGGCTACTGTTCACTTGGTCACTGCAGATGGGAGTTTT GATTGCCAAGGAAACCCAGGTGAACAAGAAGCTTTAGTTTCTTCTTTGCATTACTGTGAAGTTGTCACTG CTCTGACCACTCTTGGAAACGGTGGCTCTTTTGTTCTAAAGATGTTTACTATGTTTGAACATTGTTCCAT AAACTTGATGTACCTGCTAAACTGTTGTTTTGACCAAGTCCATGTTTTCAAACCTGCTACTAGCAAGGCA 40 GGAAACTCCGAAGTCTATGTGGTTTGCCTCCACTATAAGGGGAGAGAGGCCATCCATCCTCTGTTATCTA AGATGACCTTGAATTTTGGGACTGAAATGAAAAGGAAAGCCCTTTTTCCCCATCATGTGATTCCTGATTC 15 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TTTTCTTAAGAGACATGAAGAATGTTGTGTGTTCTTTCATAAATATCAGCTAGAGACTATTTCTGAAAAC ATTCGTCTATTTGAGTGCATGGGAAAGGCGGAACAAGAAAAGCTGAATAATTTAAGGGATTGTGCTATAC AATATTTTATGCAAAAATTTCAACTGAAACATCTTTCCAGAAATAATTGGCTAGTAAAAAAATCTAGTAT TGGTTGTAGTACAAATACAAAATGGTTTGGGCAGAGGAACAAATATTTTAAAACTTATAATGAAAGGAAG 5 ATGCTAGAAGCCCTTTCATGGAAAGATAAAGTAGCCAAAGGATACTTTAATAGTTGGGCTGAAGAACATG GTGTATATCATCCTGGGCAGAGTTCTATTTTAGAAGGAACAGCTTCCAATCTTGAGTGTCACTTATGGCA TATTTTGGAGGGAAAGAAACTGCCAAAGGTAAAATGTTCTCCTTTTTGCAATGGTGAAATTTTAAAAACT CTTAATGAAGCAATTGAAAAGTCATTAGGAGGAGCTTTTAATTTGGATTCCAAGTTTAGGCCAAAACAGC AGTATTCTTGTTCTTGTCATGTTTTTTCTGAAGAACTGATATTTTCCGAGTTGTGTAGCCTTACTGAGTG 10 CCTTCAGGATGAGCAGGTTGTAGTACCCAGCAATCAAATAAAGTGCCTGCTGGTGGGCTTTTCGACTCTC CGTAATATCAAAATGCATATACCGTTGGAAGTTCGACTCCTAGAATCAGCTGAACTCACAACTTTTAGCT GTTCATTGCTTCATGATGGAGATCCAACTTACCAGCGTTTATTTTTGGACTGCCTTCTACATTCATTGCG GGAGCTTCATACAGGAGATGTTATGATTTTGCCTGTACTTTCTTGCTTCACAAGATTTATGGCTGGTTTG ATCTTTGTACTCCACAGTTGTTTTAGATTCATCACTTTTGTTTGTCCCACATCCTCTGATCCCCTGAGGA 15 CCTGCGCAGTCCTGCTATGTGTTGGTTATCAGGACCTTCCAAATCCAGTTTTCCGATATTTGCAGAGTGT GAATGAATTGTTGAGCACTTTGCTCAACTCTGACTCACCCCAGCAGGTTTTACAGTTTGTGCCAATGGAG GTACTCCTTAAGGGGGCCCTGCTTGATTTTTTGTGGGATTTGAATGCTGCCATTGCTAAAAGGCATTTGC ATTTCATTATTCAAAGAGAGAGAGAAGAAATTATCAACAGCCTTCAGTTACAAAACTGAACATATGCTTT CTGAGATTCAACTTTATGATTTCTTATAATTTGCCCAGTATTTGCATCCTGTTGCTCTATTAATTTAAAA 20 ACCTTTTATTTTGGGGAAAGGCCAACATTTGCATCATTCAAAGTCTCATTAATTCTGGAAAACCATCCAT TCTGATCTCTAGGGTATATACACCCACAGGCATAGAGCTCTTCCACGTGGTGGAATCTATGCAATGATAG ATATTCACACTCTAAATATGAGGTGTGTGTATGTGTATGGGTGGCCACAGCCATGCTTACCTATGCCATT TAGTTGGTCTTACTTAATCTGCTTAAGATTTGCATCTGTGTACCTTTGTTCAGATTAGTTTTTTTTTTCC AGCCGATTTCCTCTTAGTGGCTAATGCTGTTAGTGAATTTTCCAACTAATTTCCTCTCATTGGTTAATGT 25 TGTTAATGAATTGAGAGAGGTAATTGAGGAAAGGAAATGAGTAAATCACTGTTCAGCAACACTGATTTCC GTTAACACATCAGTTATGAATTTCAGGGAATTCATCTCGCCAGATTCTTGATAACATGCCATTCATTGCC CTTAGGTGATTGACCCTATTTTCTTACATGGCTCAAATAAAACTAGTATGCTGTTGTATGAATCTTTTAC TGACCACACCATCCAACTATAAAAATATAACGGGACAGCTTTAAACCAAAGATCATGTTTAGAACAATGA AAAATTATTTGTTGTATCTAATACACGCCTGTATTGTGAAAAGCTTCATTTAGCAATGATGTAATAATTT 30 TTAACTTCCAGGAAATAATCTGTGAATGGAAAGATTTTTTAAGATTTTGAGATAGTGTTTAGTCTCATGT TGGGAACACATGAATGTGATGAACATAGTGAATACTAAAGAAAACGCTTCAGACTTTCAGAATGATGGTT CAGAATTTAAAATTTTTAATCTTTTCTAATTTCTTTTTTTCAGTGTGAAAATAGCACTTTACCAAAAGAT TAGCCATGAAATGGTTATTTTGCCAGTTACATTTGATTTCTTTTGTATCTGCAATGTAATGAGTTATTTT ATTTCTTCTGTATTTGCAGTGTAATGAGTTTTTGTGGCAAAGTGTATTAAGCAATTTTTCATTATCTTGA 35 AGTTCCACAAAGTGGAGAATATTTATATTCTCACATGCATTTTAGGCACTTTTGATATGTGAAAATAGAT GTATTTTCTGATGCATTTGGTTAATAAATATTAATCTGAACATTTTCATGTTCTTTGCTATTTTGAATTC CATTATAGATTCATGAATAAAGTCATTACTAGAGAGATTTTGTGTTCATCTTTTTTAAATGGAATTATGG GAGAAGTTAAAAATATAAGTTGGAAACAGACATTTTTAAAGGGAGTTTTGAATGATAGTTAATGTTCTTT CATTTTCTCTGATTTGCATTCATTAAAATTTAGTCCTTAATATTGTAATACTTATGGAAGGCCTACTTCA 40 GGAAAGAAATTGAATACTTAAGGGGGATTGGAAGAGGAGTGTGTGGAGGCCTGGAAGTAGATTTTTGAAA GAAATCTGATTTCCTACTGGTGACTCTATGGAAGCAATTCCAGTTGTATGACGATGGAAGTCTGACAGAA 16 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GGTTGATTACCAGGAGGAAAAAAAAAAACACTCTTGGAAAACCATTTTGCACTTAATAGTCTTTATGCCT TGCTAAGTGGGATTCAATATACTAATAATGCAGCTTATTTTTTACTTATTTTAACTATTTGATAAGAATG AAGTTGAAGGCATGGGTCCAGATTCTAGCATTATTACTGTCTTTAAGCCCTTCAAATAAGTATTGAATGC TTAGTGTTTTGTTGACACTAATCTGTACTGGAATATATAACACTGAAGTATTCTCAAATGTATTTTGCTT 5 TTTAACGTGGGTTTTGGAATCAATTGCTCATCTGATTCATTGGATTCAAATTGCTTTAGAGGTTAGACAT GACTTGAGGAAGGTTCAGATTGAACTGGATATATTAATAGATGACAGGTACTAATGATTTATATGAGTTT CTCTTTAAGAGAAAGGAGAGGAAAAATGCTATTCAAAAGATGAATGGAAAAGTAGAAGAAGACAGGAAGA GGTGAGAGTATGCATGTTTTACCAAGGTAGACTGTTCTTAAAAACTTTTTTCCACAGGTTTTGAGATTTT TAATTTGTAATATCTATTAAAAAACAAACATCAAA 10 >NM_001324379.2 Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 6, mRNA GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGGTGGGACTGGCTTCCCGGT GCCGCGAGGGCGGGTCCGGACAGCCTTCCCCCCAGTCCGGCGCACCATCTCCCTGCCTTGTGGCTGGAGG 15 CGCCGCGGACCCAAAGGGAGGGACCATCCCGGGAAGCAGCCCCGAGAGCGGAAGTGCAGAATGGCTTCCT CGAGAGAGTAAAGTGCAGCCTCTCCAGACACTGGGGCCCCAGTGGGCGTGGGCGAAGATTTTAATTTGAT TTGAAAATGAGTAAGTGCAGAAAGACACCAGTTCAGCAGCTAGCAAGTCCCGCGTCATTCAGCCCAGATA TTCTTGCTGACATTTTTGAACTCTTTGCCAAGAACTTTTCTTATGGCAAGCCACTTAATAATGAGTGGCA GTTACCAGATCCCAGTGAGATTTTCACCTGTGACCACACTGAACTTAATGCATTTCTTGATTTGAAGAAC 20 TCCCTAAATGAAGTAAAAAACCTACTGAGTGATAAGAAACTGGATGAGTGGCATGAGCACACTGCTTTCA CTAATAAAGCGGGGAAAATCATTTCTCATGTTAGAAAATCTGTGAATGCTGAACTTTGTACTCAAGCATG GTGTAAGTTCCATGAGATTTTGTGCAGCTTTCCACTTATTCCACAGGAAGCTTTTCAGAATGGAAAACTG AATTCTCTACACCTTTGTGAAGCTCCAGGAGCTTTTATAGCTAGTCTCAACCACTACTTAAAATCCCATC GGTTTCCTTGTCATTGGAGTTGGGTAGCGAATACTCTGAATCCATACCATGAAGCAAATGACGACCTCAT 25 GATGATTATGGATGACCGGCTTATTGCAAATACCTTGCACTGGTGGTACTTTGGTCCAGATAACACTGGT GATATCATGACCCTGAAATTCTTGACTGGACTTCAGAATTTCATAAGCAGCATGGCTACTGTTCACTTGG TCACTGCAGATGGGAGTTTTGATTGCCAAGGAAACCCAGGTGAACAAGAAGCTTTAGTTTCTTCTTTGCA TTACTGTGAAGTTGTCACTGCTCTGACCACTCTTGGAAACGGTGGCTCTTTTGTTCTAAAGATGTTTACT ATGTTTGAACATTGTTCCATAAACTTGATGTACCTGCTAAACTGTTGTTTTGACCAAGTCCATGTTTTCA 30 AACCTGCTACTAGCAAGGCAGGAAACTCCGAAGTCTATGTGGTTTGCCTCCACTATAAGGGGAGAGAGGC CATCCATCCTCTGTTATCTAAGATGACCTTGAATTTTGGGACTGAAATGAAAAGGAAAGCCCTTTTTCCC CATCATGTGATTCCTGATTCTTTTCTTAAGAGACATGAAGAATGTTGTGTGTTCTTTCATAAATATCAGC TAGAGACTATTTCTGAAAACATTCGTCTATTTGAGTGCATGGGAAAGGCGGAACAAGAAAAGCTGAATAA TTTAAGGGATTGTGCTATACAATATTTTATGCAAAAATTTCAACTGAAACATCTTTCCAGAAATAATTGG 35 CTAGTAAAAAAATCTAGTATTGGTTGTAGTACAAATACAAAATGGTTTGGGCAGAGGAACAAATATTTTA AAACTTATAATGAAAGGAAGATGCTAGAAGCCCTTTCATGGAAAGATAAAGTAGCCAAAGGATACTTTAA TAGTTGGGCTGAAGAACATGGTGTATATCATCCTGGGCAGAGTTCTATTTTAGAAGGAACAGCTTCCAAT CTTGAGTGTCACTTATGGCATATTTTGGAGGGAAAGAAACTGCCAAAGGTAAAATGTTCTCCTTTTTGCA ATGGTGAAATTTTAAAAACTCTTAATGAAGCAATTGAAAAGTCATTAGGAGGAGCTTTTAATTTGGATTC 40 CAAGTTTAGGCCAAAACAGCAGTATTCTTGTTCTTGTCATGTTTTTTCTGAAGAACTGATATTTTCCGAG TTGTGTAGCCTTACTGAGTGCCTTCAGGATGAGCAGGTTGTAGTACCCAGCAATCAAATAAAGTGCCTGC 17 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGGTGGGCTTTTCGACTCTCCGTAATATCAAAATGCATATACCGTTGGAAGTTCGACTCCTAGAATCAGC TGAACTCACAACTTTTAGCTGTTCATTGCTTCATGATGGAGATCCAACTTACCAGCGTTTATTTTTGGAC TGCCTTCTACATTCATTGCGGGAGCTTCATACAGGAGATGTTATGATTTTGCCTGTACTTTCTTGCTTCA CAAGATTTATGGCTGGTTTGATCTTTGTACTCCACAGTTGTTTTAGATTCATCACTTTTGTTTGTCCCAC 5 ATCCTCTGATCCCCTGAGGACCTGCGCAGTCCTGCTATGTGTTGGTTATCAGGACCTTCCAAATCCAGTT TTCCGATATTTGCAGAGTGTGAATGAATTGTTGAGCACTTTGCTCAACTCTGACTCACCCCAGCAGGTTT TACAGTTTGTGCCAATGGAGGTACTCCTTAAGGGGGCCCTGCTTGATTTTTTGTGGGATTTGAATGCTGC CATTGCTAAAAGGCATTTGCATTTCATTATTCAAAGAGAGAGAGAAGAAATTATCAACAGCCTTCAGTTA CAAAACTGAACATATGCTTTCTGAGATTCAACTTTATGATTTCTTATAATTTGCCCAGTATTTGCATCCT 10 GTTGCTCTATTAATTTAAAAACCTTTTATTTTGGGGAAAGGCCAACATTTGCATCATTCAAAGTCTCATT AATTCTGGAAAACCATCCATTCTGATCTCTAGGGTATATACACCCACAGGCATAGAGCTCTTCCACGTGG TGGAATCTATGCAATGATAGATATTCACACTCTAAATATGAGGTGTGTGTATGTGTATGGGTGGCCACAG CCATGCTTACCTATGCCATTTAGTTGGTCTTACTTAATCTGCTTAAGATTTGCATCTGTGTACCTTTGTT CAGATTAGTTTTTTTTTTCCAGCCGATTTCCTCTTAGTGGCTAATGCTGTTAGTGAATTTTCCAACTAAT 15 TTCCTCTCATTGGTTAATGTTGTTAATGAATTGAGAGAGGTAATTGAGGAAAGGAAATGAGTAAATCACT GTTCAGCAACACTGATTTCCGTTAACACATCAGTTATGAATTTCAGGGAATTCATCTCGCCAGATTCTTG ATAACATGCCATTCATTGCCCTTAGGTGATTGACCCTATTTTCTTACATGGCTCAAATAAAACTAGTATG CTGTTGTATGAATCTTTTACTGACCACACCATCCAACTATAAAAATATAACGGGACAGCTTTAAACCAAA GATCATGTTTAGAACAATGAAAAATTATTTGTTGTATCTAATACACGCCTGTATTGTGAAAAGCTTCATT 20 TAGCAATGATGTAATAATTTTTAACTTCCAGGAAATAATCTGTGAATGGAAAGATTTTTTAAGATTTTGA GATAGTGTTTAGTCTCATGTTGGGAACACATGAATGTGATGAACATAGTGAATACTAAAGAAAACGCTTC AGACTTTCAGAATGATGGTTCAGAATTTAAAATTTTTAATCTTTTCTAATTTCTTTTTTTCAGTGTGAAA ATAGCACTTTACCAAAAGATTAGCCATGAAATGGTTATTTTGCCAGTTACATTTGATTTCTTTTGTATCT GCAATGTAATGAGTTATTTTATTTCTTCTGTATTTGCAGTGTAATGAGTTTTTGTGGCAAAGTGTATTAA 25 GCAATTTTTCATTATCTTGAAGTTCCACAAAGTGGAGAATATTTATATTCTCACATGCATTTTAGGCACT TTTGATATGTGAAAATAGATGTATTTTCTGATGCATTTGGTTAATAAATATTAATCTGAACATTTTCATG TTCTTTGCTATTTTGAATTCCATTATAGATTCATGAATAAAGTCATTACTAGAGAGATTTTGTGTTCATC TTTTTTAAATGGAATTATGGGAGAAGTTAAAAATATAAGTTGGAAACAGACATTTTTAAAGGGAGTTTTG AATGATAGTTAATGTTCTTTCATTTTCTCTGATTTGCATTCATTAAAATTTAGTCCTTAATATTGTAATA 30 CTTATGGAAGGCCTACTTCAGGAAAGAAATTGAATACTTAAGGGGGATTGGAAGAGGAGTGTGTGGAGGC CTGGAAGTAGATTTTTGAAAGAAATCTGATTTCCTACTGGTGACTCTATGGAAGCAATTCCAGTTGTATG ACGATGGAAGTCTGACAGAAGGTTGATTACCAGGAGGAAAAAAAAAAACACTCTTGGAAAACCATTTTGC ACTTAATAGTCTTTATGCCTTGCTAAGTGGGATTCAATATACTAATAATGCAGCTTATTTTTTACTTATT TTAACTATTTGATAAGAATGAAGTTGAAGGCATGGGTCCAGATTCTAGCATTATTACTGTCTTTAAGCCC 35 TTCAAATAAGTATTGAATGCTTAGTGTTTTGTTGACACTAATCTGTACTGGAATATATAACACTGAAGTA TTCTCAAATGTATTTTGCTTTTTAACGTGGGTTTTGGAATCAATTGCTCATCTGATTCATTGGATTCAAA TTGCTTTAGAGGTTAGACATGACTTGAGGAAGGTTCAGATTGAACTGGATATATTAATAGATGACAGGTA CTAATGATTTATATGAGTTTCTCTTTAAGAGAAAGGAGAGGAAAAATGCTATTCAAAAGATGAATGGAAA AGTAGAAGAAGACAGGAAGAGGTGAGAGTATGCATGTTTTACCAAGGTAGACTGTTCTTAAAAACTTTTT 40 TCCACAGGTTTTGAGATTTTTAATTTGTAATATCTATTAAAAAACAAACATCAAA 18 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_018348.6 Homo sapiens cap methyltransferase 2 (CMTR2), transcript variant 1, mRNA GAGTGCCTCCTGGTCCCTGTCTGCCGGCATTCGCGGCTGCGGGGCCCGGAGGTGGGACTGGCTTCCCGGT GCCGCGAGGGCGGGTCCGGACAGCCTTCCCCCCAGTCCGGCGCACCATCTCCCTGCCTTGTGGCTGGAGG 5 CGCCGCGGACCCAAAGGGAGGGACCATCCCGGGAAGCAGCCCCGAGAGCGGAAGTGCAGAATGGCTTCCT CGAGAGAGTAAAGTGCAGCCTCTCCAGACACTGGGGCCCCAGTGGGCGTGGGCGAAGTTGCTGGTAGGAG GAGTTGGCGGAAGCACTTGGAACTCCTTTATAAGTGTCAGCTGTGAGATTTTAATTTGATTTGAAAATGA GTAAGTGCAGAAAGACACCAGTTCAGCAGCTAGCAAGTCCCGCGTCATTCAGCCCAGATATTCTTGCTGA CATTTTTGAACTCTTTGCCAAGAACTTTTCTTATGGCAAGCCACTTAATAATGAGTGGCAGTTACCAGAT 10 CCCAGTGAGATTTTCACCTGTGACCACACTGAACTTAATGCATTTCTTGATTTGAAGAACTCCCTAAATG AAGTAAAAAACCTACTGAGTGATAAGAAACTGGATGAGTGGCATGAGCACACTGCTTTCACTAATAAAGC GGGGAAAATCATTTCTCATGTTAGAAAATCTGTGAATGCTGAACTTTGTACTCAAGCATGGTGTAAGTTC CATGAGATTTTGTGCAGCTTTCCACTTATTCCACAGGAAGCTTTTCAGAATGGAAAACTGAATTCTCTAC ACCTTTGTGAAGCTCCAGGAGCTTTTATAGCTAGTCTCAACCACTACTTAAAATCCCATCGGTTTCCTTG 15 TCATTGGAGTTGGGTAGCGAATACTCTGAATCCATACCATGAAGCAAATGACGACCTCATGATGATTATG GATGACCGGCTTATTGCAAATACCTTGCACTGGTGGTACTTTGGTCCAGATAACACTGGTGATATCATGA CCCTGAAATTCTTGACTGGACTTCAGAATTTCATAAGCAGCATGGCTACTGTTCACTTGGTCACTGCAGA TGGGAGTTTTGATTGCCAAGGAAACCCAGGTGAACAAGAAGCTTTAGTTTCTTCTTTGCATTACTGTGAA GTTGTCACTGCTCTGACCACTCTTGGAAACGGTGGCTCTTTTGTTCTAAAGATGTTTACTATGTTTGAAC 20 ATTGTTCCATAAACTTGATGTACCTGCTAAACTGTTGTTTTGACCAAGTCCATGTTTTCAAACCTGCTAC TAGCAAGGCAGGAAACTCCGAAGTCTATGTGGTTTGCCTCCACTATAAGGGGAGAGAGGCCATCCATCCT CTGTTATCTAAGATGACCTTGAATTTTGGGACTGAAATGAAAAGGAAAGCCCTTTTTCCCCATCATGTGA TTCCTGATTCTTTTCTTAAGAGACATGAAGAATGTTGTGTGTTCTTTCATAAATATCAGCTAGAGACTAT TTCTGAAAACATTCGTCTATTTGAGTGCATGGGAAAGGCGGAACAAGAAAAGCTGAATAATTTAAGGGAT 25 TGTGCTATACAATATTTTATGCAAAAATTTCAACTGAAACATCTTTCCAGAAATAATTGGCTAGTAAAAA AATCTAGTATTGGTTGTAGTACAAATACAAAATGGTTTGGGCAGAGGAACAAATATTTTAAAACTTATAA TGAAAGGAAGATGCTAGAAGCCCTTTCATGGAAAGATAAAGTAGCCAAAGGATACTTTAATAGTTGGGCT GAAGAACATGGTGTATATCATCCTGGGCAGAGTTCTATTTTAGAAGGAACAGCTTCCAATCTTGAGTGTC ACTTATGGCATATTTTGGAGGGAAAGAAACTGCCAAAGGTAAAATGTTCTCCTTTTTGCAATGGTGAAAT 30 TTTAAAAACTCTTAATGAAGCAATTGAAAAGTCATTAGGAGGAGCTTTTAATTTGGATTCCAAGTTTAGG CCAAAACAGCAGTATTCTTGTTCTTGTCATGTTTTTTCTGAAGAACTGATATTTTCCGAGTTGTGTAGCC TTACTGAGTGCCTTCAGGATGAGCAGGTTGTAGTACCCAGCAATCAAATAAAGTGCCTGCTGGTGGGCTT TTCGACTCTCCGTAATATCAAAATGCATATACCGTTGGAAGTTCGACTCCTAGAATCAGCTGAACTCACA ACTTTTAGCTGTTCATTGCTTCATGATGGAGATCCAACTTACCAGCGTTTATTTTTGGACTGCCTTCTAC 35 ATTCATTGCGGGAGCTTCATACAGGAGATGTTATGATTTTGCCTGTACTTTCTTGCTTCACAAGATTTAT GGCTGGTTTGATCTTTGTACTCCACAGTTGTTTTAGATTCATCACTTTTGTTTGTCCCACATCCTCTGAT CCCCTGAGGACCTGCGCAGTCCTGCTATGTGTTGGTTATCAGGACCTTCCAAATCCAGTTTTCCGATATT TGCAGAGTGTGAATGAATTGTTGAGCACTTTGCTCAACTCTGACTCACCCCAGCAGGTTTTACAGTTTGT GCCAATGGAGGTACTCCTTAAGGGGGCCCTGCTTGATTTTTTGTGGGATTTGAATGCTGCCATTGCTAAA 40 AGGCATTTGCATTTCATTATTCAAAGAGAGAGAGAAGAAATTATCAACAGCCTTCAGTTACAAAACTGAA CATATGCTTTCTGAGATTCAACTTTATGATTTCTTATAATTTGCCCAGTATTTGCATCCTGTTGCTCTAT 19 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TAATTTAAAAACCTTTTATTTTGGGGAAAGGCCAACATTTGCATCATTCAAAGTCTCATTAATTCTGGAA AACCATCCATTCTGATCTCTAGGGTATATACACCCACAGGCATAGAGCTCTTCCACGTGGTGGAATCTAT GCAATGATAGATATTCACACTCTAAATATGAGGTGTGTGTATGTGTATGGGTGGCCACAGCCATGCTTAC CTATGCCATTTAGTTGGTCTTACTTAATCTGCTTAAGATTTGCATCTGTGTACCTTTGTTCAGATTAGTT 5 TTTTTTTTCCAGCCGATTTCCTCTTAGTGGCTAATGCTGTTAGTGAATTTTCCAACTAATTTCCTCTCAT TGGTTAATGTTGTTAATGAATTGAGAGAGGTAATTGAGGAAAGGAAATGAGTAAATCACTGTTCAGCAAC ACTGATTTCCGTTAACACATCAGTTATGAATTTCAGGGAATTCATCTCGCCAGATTCTTGATAACATGCC ATTCATTGCCCTTAGGTGATTGACCCTATTTTCTTACATGGCTCAAATAAAACTAGTATGCTGTTGTATG AATCTTTTACTGACCACACCATCCAACTATAAAAATATAACGGGACAGCTTTAAACCAAAGATCATGTTT 10 AGAACAATGAAAAATTATTTGTTGTATCTAATACACGCCTGTATTGTGAAAAGCTTCATTTAGCAATGAT GTAATAATTTTTAACTTCCAGGAAATAATCTGTGAATGGAAAGATTTTTTAAGATTTTGAGATAGTGTTT AGTCTCATGTTGGGAACACATGAATGTGATGAACATAGTGAATACTAAAGAAAACGCTTCAGACTTTCAG AATGATGGTTCAGAATTTAAAATTTTTAATCTTTTCTAATTTCTTTTTTTCAGTGTGAAAATAGCACTTT ACCAAAAGATTAGCCATGAAATGGTTATTTTGCCAGTTACATTTGATTTCTTTTGTATCTGCAATGTAAT 15 GAGTTATTTTATTTCTTCTGTATTTGCAGTGTAATGAGTTTTTGTGGCAAAGTGTATTAAGCAATTTTTC ATTATCTTGAAGTTCCACAAAGTGGAGAATATTTATATTCTCACATGCATTTTAGGCACTTTTGATATGT GAAAATAGATGTATTTTCTGATGCATTTGGTTAATAAATATTAATCTGAACATTTTCATGTTCTTTGCTA TTTTGAATTCCATTATAGATTCATGAATAAAGTCATTACTAGAGAGATTTTGTGTTCATCTTTTTTAAAT GGAATTATGGGAGAAGTTAAAAATATAAGTTGGAAACAGACATTTTTAAAGGGAGTTTTGAATGATAGTT 20 AATGTTCTTTCATTTTCTCTGATTTGCATTCATTAAAATTTAGTCCTTAATATTGTAATACTTATGGAAG GCCTACTTCAGGAAAGAAATTGAATACTTAAGGGGGATTGGAAGAGGAGTGTGTGGAGGCCTGGAAGTAG ATTTTTGAAAGAAATCTGATTTCCTACTGGTGACTCTATGGAAGCAATTCCAGTTGTATGACGATGGAAG TCTGACAGAAGGTTGATTACCAGGAGGAAAAAAAAAAACACTCTTGGAAAACCATTTTGCACTTAATAGT CTTTATGCCTTGCTAAGTGGGATTCAATATACTAATAATGCAGCTTATTTTTTACTTATTTTAACTATTT 25 GATAAGAATGAAGTTGAAGGCATGGGTCCAGATTCTAGCATTATTACTGTCTTTAAGCCCTTCAAATAAG TATTGAATGCTTAGTGTTTTGTTGACACTAATCTGTACTGGAATATATAACACTGAAGTATTCTCAAATG TATTTTGCTTTTTAACGTGGGTTTTGGAATCAATTGCTCATCTGATTCATTGGATTCAAATTGCTTTAGA GGTTAGACATGACTTGAGGAAGGTTCAGATTGAACTGGATATATTAATAGATGACAGGTACTAATGATTT ATATGAGTTTCTCTTTAAGAGAAAGGAGAGGAAAAATGCTATTCAAAAGATGAATGGAAAAGTAGAAGAA 30 GACAGGAAGAGGTGAGAGTATGCATGTTTTACCAAGGTAGACTGTTCTTAAAAACTTTTTTCCACAGGTT TTGAGATTTTTAATTTGTAATATCTATTAAAAAACAAACATCAAA In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and 35 the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. By “decreases” is meant a reduction by at least about 5% relative to a reference level. A 20 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages. As used herein, the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term 5 “determining” is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase “determining a level” of an analyte or “detecting” an analyte is used. “Detect” refers to identifying the presence, absence or amount of the analyte to be 10 detected. In some embodiments, the analyte is a modified nucleobase or modified polynucleotide (e.g., RNA) comprising a 2’ O-methlyl modification. By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, 15 metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. The term “expression” or “expressed” as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA 20 that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable 25 expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. The disclosure provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, 30 the methods of the disclosure provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the disclosure provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity. 21 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 By “fibrillarin polypeptide” or “FBL polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_001427.2, or a fragment thereof, and having methyltransferase or preribosomal RNA processing activity. The sequence of an exemplary FBL polypeptide is provided below: 5 >NP_001427.2 rRNA 2'-O-methyltransferase fibrillarin [Homo sapiens] MKPGFSPRGGGFGGRGGFGDRGGRGGRGGFGGGRGRGGGFRGRGRGGGGGGGGGGGGGRGGGGFHSGGNR GRGRGGKRGNQSGKNVMVEPHRHEGVFICRGKEDALVTKNLVPGESVYGEKRVSISEGDDKIEYRAWNPF RSKLAAAILGGVDQIHIKPGAKVLYLGAASGTTVSHVSDIVGPDGLVYAVEFSHRSGRDLINLAKKRTNI IPVIEDARHPHKYRMLIAMVDVIFADVAQPDQTRIVALNAHTFLRNGGHFVISIKANCIDSTASAEAVFA 10 SEVKKMQQENMKPQEQLTLEPYERDHAVVVGVYRPPPKVKN By “fibrillarin polynucleotide” or “FBL polynucleotide” is meant a nucleic acid molecule encoding an FBL polypeptide or fragment thereof. The sequence of an exemplary FBL polynucleotide is provided below: 15 >NM_001436.4 Homo sapiens fibrillarin (FBL), mRNA CTTTTCCACGTGCGAAAGCCCCGGACTCGTGGAGTTGTGAACGCCGCGGACTCCGGAGCCGCACAAACCA GGGCTCGCCATGAAGCCAGGATTCAGTCCCCGTGGGGGTGGCTTTGGCGGCCGAGGGGGCTTTGGTGACC GTGGTGGTCGTGGAGGCCGAGGGGGCTTTGGCGGGGGCCGAGGTCGAGGCGGAGGCTTTAGAGGTCGTGG ACGAGGAGGAGGTGGAGGCGGCGGCGGCGGTGGAGGAGGAGGAAGAGGTGGTGGAGGCTTCCATTCTGGT 20 GGCAACCGGGGTCGTGGTCGGGGAGGAAAAAGAGGAAACCAGTCGGGGAAGAATGTGATGGTGGAGCCGC ATCGGCATGAGGGTGTCTTCATTTGTCGAGGAAAGGAAGATGCACTGGTCACCAAGAACCTGGTCCCTGG GGAATCAGTTTATGGAGAGAAGAGAGTCTCGATTTCGGAAGGAGATGACAAAATTGAGTACCGAGCCTGG AACCCCTTCCGCTCCAAGCTAGCAGCAGCAATCCTGGGTGGTGTGGACCAGATCCACATCAAACCGGGGG CTAAGGTTCTCTACCTCGGGGCTGCCTCGGGCACCACGGTCTCCCATGTCTCTGACATCGTTGGTCCGGA 25 TGGTCTAGTCTATGCAGTCGAGTTCTCCCACCGCTCTGGCCGTGACCTCATTAACTTGGCCAAGAAGAGG ACCAACATCATTCCTGTGATCGAGGATGCTCGACACCCACACAAATACCGCATGCTCATCGCAATGGTGG ATGTGATCTTTGCTGATGTGGCCCAGCCAGACCAGACCCGGATTGTGGCCCTGAATGCCCACACCTTCCT GCGTAATGGAGGACACTTTGTGATTTCCATTAAGGCCAACTGCATTGACTCCACAGCCTCAGCCGAGGCC GTGTTTGCCTCCGAAGTGAAAAAGATGCAACAGGAGAACATGAAGCCGCAGGAGCAGTTGACCCTTGAGC 30 CATATGAAAGAGACCATGCCGTGGTCGTGGGAGTGTACAGGCCACCCCCCAAGGTGAAGAACTGAAGTTC AGCGCTGTCAGGATTGCGAGAGATGTGTGTTGATACTGTTGCACGTGTGTTTTTCTATTAAAAGACTCAT CCGTC By “fibrillarin like 1 polypeptide” or “FBLL1 polypeptide” is meant a polypeptide 35 having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_001342203.1, or a fragment thereof, and having methyltransferase activity. The sequence of an exemplary FBLL1 polypeptide is provided below: 22 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_001342203.1 rRNA/tRNA 2'-O-methyltransferase fibrillarin-like protein 1 [Homo sapiens] MKSAASSRGGGGGGRGGGGWGSWGGGRGGGGGAGKGGGGDGGGQGGKGGFGARARGFGGGGRGRGRGGGD GKDRGGGGQRRGGVAKSKSRRRKGAMVVSVEPHRHEGVFIYRGAEDALVTLNMVPGQSVYGERRVTVTEG 5 GVKQEYRTWNPFRSKLAAAILGGVDQIHIKPKSKVLYLGAASGTTVSHVSDIIGPDGLVYAVEFSHRAGR DLVNVAKKRTNIIPVLEDARHPLKYRMLIGMVDVIFADVAQPDQSRIVALNAHTFLRNGGHFLISIKANC IDSTASAEAVFASEVRKLQQENLKPQEQLTLEPYERDHAVVVGVYRPLPKSSSK By “fibrillarin like 1 polynucleotide” or “FBLL1 polynucleotide” is meant a nucleic acid 10 molecule encoding an FBLL1 polypeptide or fragment thereof. The sequence of an exemplary FBLL1 polynucleotide is provided below: >NM_001355274.2 Homo sapiens fibrillarin like 1 (FBLL1), mRNA ACAGCCGCTGCGTCAAGGGGCGGCTCCGGGATGCGGGCACTGCGGTAGAGCCCGTCCTGCCCCCGCCGCC CGCCGCCCGCCGCCCGCCTCCGCTGCTAAACCCGCGGACAACCGCCGGGACGACCACCCAGAGCCACCAG 15 AGCCCCCTGACACCCGCCCCGAGGCACCGGTAACCCACGGCACCCGCCCCGGTGCGTGCCATGAAGTCGG CCGCGAGCTCGCGCGGGGGCGGTGGGGGCGGCCGCGGGGGCGGCGGCTGGGGCAGCTGGGGCGGGGGCCG AGGCGGCGGCGGCGGCGCGGGCAAGGGCGGCGGGGGCGACGGCGGCGGCCAGGGGGGCAAGGGCGGCTTC GGGGCGCGGGCGCGCGGCTTCGGCGGGGGCGGCCGGGGCCGGGGGCGCGGCGGCGGCGACGGCAAGGATC GCGGCGGCGGTGGACAGCGGCGGGGCGGCGTGGCCAAGAGCAAGAGCCGCCGCAGGAAGGGCGCCATGGT 20 GGTGTCGGTGGAGCCGCACCGGCACGAGGGCGTCTTCATCTACCGCGGGGCGGAGGACGCGCTGGTCACG CTGAACATGGTGCCGGGCCAGTCTGTGTACGGCGAGAGGCGCGTCACGGTGACCGAGGGCGGCGTGAAGC AGGAGTACCGCACGTGGAACCCGTTCCGCTCTAAGCTGGCCGCGGCCATCCTGGGCGGGGTGGACCAGAT CCACATCAAGCCCAAGTCCAAGGTGCTGTACCTGGGCGCCGCGTCGGGCACCACCGTCTCCCATGTCTCC GACATCATTGGCCCAGACGGCCTGGTCTACGCCGTCGAGTTCTCCCACCGCGCCGGCCGCGATCTGGTCA 25 ACGTGGCCAAGAAGCGCACCAACATCATTCCGGTCCTGGAGGACGCGCGGCACCCGCTCAAGTACCGCAT GCTCATCGGGATGGTGGACGTGATCTTCGCCGACGTGGCCCAGCCGGACCAGTCCCGCATCGTGGCCCTG AACGCCCACACCTTCCTGCGCAATGGGGGCCACTTTCTCATCTCCATCAAGGCCAACTGCATCGACTCCA CCGCATCCGCCGAGGCTGTGTTTGCTTCTGAGGTGAGGAAGTTGCAGCAGGAGAACTTGAAGCCTCAAGA GCAGCTGACCCTGGAGCCCTATGAGCGGGACCACGCTGTGGTGGTCGGGGTCTACCGACCTCTTCCCAAG 30 AGCAGCAGCAAATAGCACCCAGCTCAGGCTCGCCTGCCATCTCCCCAAGGCTGCGTTGTGTTTGCTATTA TTTTCTGTGTGTTTTCTTTGTGAGTGTTTTGTTTTGTTGTTTTTCTATTAAACTGCATAAAGAAACGGCA By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the 35 reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. By “FtsJ RNA 2'-O-methyltransferase 1 polypeptide” or “FTSJ1 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001269086.1, NP_036412.1, or NP_803188.1, or a fragment thereof, and having 23 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 methyltransferase activity. The sequences of exemplary FTSJ1 polypeptides are provided below: >NP_001269086.1 tRNA (cytidine(32)/guanosine(34)-2'-O)-methyltransferase isoform c [Homo sapiens] 5 MALNIATHVLKPGGCFVAKIFRGRDVTLLYSQLQVFFSSVLCAKPRSSRNSSIEAFAVCQGYDPPEGFIP DLSKPLLDHSYDPDFNQLDGPTRIIVPFVTCGDLSSYDSDRSYPLDLEGGSEYKYTPPTQPPISPPYQEA CTLKRKGQLAKEIRPQDCPISRVDTFPQPLAAPQCHTLLAPEMEDNEMSCSP >NP_036412.1 tRNA (cytidine(32)/guanosine(34)-2'-O)-methyltransferase isoform 10 a [Homo sapiens] MGRTSKDKRDVYYRLAKENGWRARSAFKLLQLDKEFQLFQGVTRAVDLCAAPGSWSQVLSQKIGGQGSGH VVAVDLQAMAPLPGVVQIQGDITQLSTAKEIIQHFKGCPADLVVCDGAPDVTGLHDVDEYMQAQLLLAAL NIATHVLKPGGCFVAKIFRGRDVTLLYSQLQVFFSSVLCAKPRSSRNSSIEAFAVCQGYDPPEGFIPDLS KPLLDHSYDPDFNQLDGPTRIIVPFVTCGDLSSYDSDRSYPLDLEGGSEYKYTPPTQPPISPPYQEACTL 15 KRKGQLAKEIRPQDCPISRVDTFPQPLAAPQCHTLLAPEMEDNEMSCSP >NP_803188.1 tRNA (cytidine(32)/guanosine(34)-2'-O)-methyltransferase isoform b [Homo sapiens] MGRTSKDKRDVYYRLAKENGWRARSAFKLLQLDKEFQLFQGVTRAVDLCAAPGSWSQVLSQKIGGQGSGH 20 VVAVDLQAMAPLPGVVQIQGDITQLSTAKEIIQHFKGCPADLVVCDGAPDVTGLHDVDEYMQAQLLLAAL NIATHVLKPGGCFVAKIFRGRDVTLLYSQLQVFFSSVLCAKPRSSRNSSIEAFAVCQGYDPPEGFIPDLS KPLLDHSYDFNQLDGPTRIIVPFVTCGDLSSYDSDRSYPLDLEGGSEYKYTPPTQPPISPPYQEACTLKR KGQLAKEIRPQDCPISRVDTFPQPLAAPQCHTLLAPEMEDNEMSCSP 25 By “FtsJ RNA 2'-O-methyltransferase 1 polynucleotide” or “FTSJ1 polynucleotide” is meant a nucleic acid molecule encoding an FTSJ1 polypeptide or fragment thereof. The sequences of exemplary FTSJ1 polynucleotides are provided below: >NM_001282157.1 Homo sapiens FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1), transcript variant 4, mRNA 30 AAATCGATCTTGGCCTCGGAAGAAAAAGAACAAAGAATCGCTGGCAACTTGCGCGTCGCGTCGCTGCAGC CACGCAGTGCGTGGATTCAGCCGCTTTGGCCGTGCCCGTTGCATGCCGGGAAACGCAGTTCGCGAGCCCC GGATACGTCGACAATAGATGACGGGGCTCACGTTGTACGTTCACATCAGGTCCCGGCCCGCCGGAACCTG GGCGATCCACGATGCCGAGTTTGCCACGCTGCGACAGCCCATAGGCTTGCCCCCCCGGGCATTCGGGTGG ACTACGAACACAAACTGAAGCCCTAGGACTTGTCGCCCGTTTGCGCTCTCGCCGAGGCACAGGCTGCTCG 35 CGGACCACCCTGCTCCGAAAACTAAGCTGTCCACTGCCAAGGAGATCATCCAGCACTTTAAGGGCTGCCC TGCGGACCTAGTGGTGTGTGACGGGGCTCCTGATGGCTCTGAACATTGCTACACATGTCCTGAAGCCAGG GGGCTGCTTTGTGGCCAAGATATTCCGAGGCCGGGATGTGACGCTCCTCTACAGCCAGCTGCAGGTCTTC TTCTCCAGCGTGCTGTGTGCCAAGCCCAGGAGCAGCCGGAACTCTAGCATCGAGGCCTTCGCTGTCTGTC AGGGCTATGACCCTCCCGAGGGCTTCATCCCGGACCTGAGCAAACCCCTGCTGGACCATTCTTACGACCC 24 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 AGATTTCAACCAGCTGGATGGTCCCACCCGCATCATTGTGCCTTTTGTGACCTGTGGGGACCTGAGCTCC TATGATTCGGACCGCAGTTACCCACTGGACCTAGAGGGCGGCTCAGAGTACAAGTACACTCCACCCACAC AGCCCCCCATCTCGCCACCATACCAGGAGGCCTGCACGTTGAAGAGGAAGGGGCAGCTGGCCAAGGAGAT CCGCCCCCAGGACTGCCCCATCAGCAGAGTGGACACGTTTCCCCAGCCCCTGGCCGCCCCTCAGTGCCAC 5 ACCCTGCTGGCCCCTGAGATGGAAGACAATGAAATGAGTTGTTCACCTTAACCCATTACGCTGGCAAACT GATACAACTCAGAAACTGCTCAATGTCAGGAAAACCGGAACTTGTTGATGAACTTGTTTTCAAAATATCA TCTCATCAACGAGGCCTGGACAAACAAGCCCTGAGGAGGGATCTGCAACAACCCTGAAGACAACAAGGAA AGAAACCATGAAAGTCTGTCTGTACTGCAGTGGGAATTCTTGAGTGAGGTCTTACCTCTTCTTTAAACCT CTTCAGGAGTGTCCTGATTATGTCCAGAATTTTCCCTAAAGGCAGGGATTCTTAACCTGGATAGAAGCCA 10 GGGGTAACCATGAACTTGATGGAAGAAAATGTTACATCTTTATTTTCAGCAATGAAACTGAAATTTAGCC TTACTCCCAAGTTATAAATGCTGGCAACAAATCACAGTAGTAAAAGCAGTACCTGGGATTTTCCCACCAA TACAAACCAGTTACAGCAACTGTAATGTAACATAAAAAGGGCATCTTGAAGTATTGTTCATATTTGTCAC CTCTTGGAATGATAGCAGATCCTGTAAGTTGATGTATTAATTAAAAAGCCCATATATTACTGCATCTCAA TTTTGTTTCAATTTAATATATAGTAATAATTGTAATTCAATGTAATTGGCTTCCATTGT 15 >NM_012280.4 Homo sapiens FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1), transcript variant 1, mRNA GTTCACATCAGGTCCCGGCCCGCCGGAACCTGGGCGATCCACGATGCCGAGTTTGCCACGCTGCGACAGC CCATAGGCTTGCCCCCCCGGGCATTCGGGTGGACTACGAACACAAACTGAAGCCCTAGGACTTGTCGCCC 20 GTTTGCGCTCTCGCCGAGGCACAGGCTGCTCGCGGACCACCCTGCTCCGAAAACTAAGAGGTAGGTGGTA GCCCATTCATCTGGTTACTGATACTGGCCGGCATCAGTGGACTGTCGGCAGGTCCTTGAGCAACTGGTGT GTGAAATGGGACGGACGTCAAAGGACAAGCGGGATGTCTACTACCGCCTGGCCAAGGAGAATGGCTGGCG TGCTCGCAGCGCCTTCAAACTGCTACAACTGGATAAGGAATTCCAACTCTTCCAAGGCGTGACACGGGCA GTTGACCTGTGTGCAGCCCCAGGCAGCTGGAGCCAGGTGCTGAGCCAGAAGATCGGGGGCCAAGGGTCCG 25 GCCACGTGGTGGCTGTGGACCTGCAGGCTATGGCTCCACTACCAGGTGTGGTACAGATCCAGGGGGACAT CACCCAGCTGTCCACTGCCAAGGAGATCATCCAGCACTTTAAGGGCTGCCCTGCGGACCTAGTGGTGTGT GACGGGGCTCCTGATGTAACCGGTCTCCATGATGTTGATGAGTATATGCAGGCCCAGCTCCTCCTAGCTG CTCTGAACATTGCTACACATGTCCTGAAGCCAGGGGGCTGCTTTGTGGCCAAGATATTCCGAGGCCGGGA TGTGACGCTCCTCTACAGCCAGCTGCAGGTCTTCTTCTCCAGCGTGCTGTGTGCCAAGCCCAGGAGCAGC 30 CGGAACTCTAGCATCGAGGCCTTCGCTGTCTGTCAGGGCTATGACCCTCCCGAGGGCTTCATCCCGGACC TGAGCAAACCCCTGCTGGACCATTCTTACGACCCAGATTTCAACCAGCTGGATGGTCCCACCCGCATCAT TGTGCCTTTTGTGACCTGTGGGGACCTGAGCTCCTATGATTCGGACCGCAGTTACCCACTGGACCTAGAG GGCGGCTCAGAGTACAAGTACACTCCACCCACACAGCCCCCCATCTCGCCACCATACCAGGAGGCCTGCA CGTTGAAGAGGAAGGGGCAGCTGGCCAAGGAGATCCGCCCCCAGGACTGCCCCATCAGCAGAGTGGACAC 35 GTTTCCCCAGCCCCTGGCCGCCCCTCAGTGCCACACCCTGCTGGCCCCTGAGATGGAAGACAATGAAATG AGTTGTTCACCTTAACCCATTACGCTGGCAAACTGATACAACTCAGAAACTGCTCAATGTCAGGAAAACC GGAACTTGTTGATGAACTTGTTTTCAAAATATCATCTCATCAACGAGGCCTGGACAAACAAGCCCTGAGG AGGGATCTGCAACAACCCTGAAGACAACAAGGAAAGAAACCATGAAAGTCTGTCTGTACTGCAGTGGGAA TTCTTGAGTGAGGTCTTACCTCTTCTTTAAACCTCTTCAGGAGTGTCCTGATTATGTCCAGAATTTTCCC 40 TAAAGGCAGGGATTCTTAACCTGGATAGAAGCCAGGGGTAACCATGAACTTGATGGAAGAAAATGTTACA TCTTTATTTTCAGCAATGAAACTGAAATTTAGCCTTACTCCCAAGTTATAAATGCTGGCAACAAATCACA 25 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GTAGTAAAAGCAGTACCTGGGATTTTCCCACCAATACAAACCAGTTACAGCAACTGTAATGTAACATAAA AAGGGCATCTTGAAGTATTGTTCATATTTGTCACCTCTTGGAATGATAGCAGATCCTGTAAGTTGATGTA TTAATTAAAAAGCCCATATATTACTGCATCTCAATTTTGTTTCAATTTAATATATAGTAATAATTGTAAT TCAATGTAA 5 >NM_177439.3 Homo sapiens FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1), transcript variant 3, mRNA GTTCACATCAGGTCCCGGCCCGCCGGAACCTGGGCGATCCACGATGCCGAGTTTGCCACGCTGCGACAGC CCATAGGCTTGCCCCCCCGGGCATTCGGGTGGACTACGAACACAAACTGAAGCCCTAGGACTTGTCGCCC 10 GTTTGCGCTCTCGCCGAGGCACAGGCTGCTCGCGGACCACCCTGCTCCGAAAACTAAGAAGGGGACCAGT AGACGGGGTGGATTCGAGGTGGGGACATGGGACGCAGCGGTCGGAGCCGCCTGGAGGGGTACCCGGTGGT CCCGATGGAGCTATCATCTTTGAGAGGTAGGTGGTAGCCCATTCATCTGGTTACTGATACTGGCCGGCAT CAGTGGACTGTCGGCAGGTCCTTGAGCAACTGGTGTGTGAAATGGGACGGACGTCAAAGGACAAGCGGGA TGTCTACTACCGCCTGGCCAAGGAGAATGGCTGGCGTGCTCGCAGCGCCTTCAAACTGCTACAACTGGAT 15 AAGGAATTCCAACTCTTCCAAGGCGTGACACGGGCAGTTGACCTGTGTGCAGCCCCAGGCAGCTGGAGCC AGGTGCTGAGCCAGAAGATCGGGGGCCAAGGGTCCGGCCACGTGGTGGCTGTGGACCTGCAGGCTATGGC TCCACTACCAGGTGTGGTACAGATCCAGGGGGACATCACCCAGCTGTCCACTGCCAAGGAGATCATCCAG CACTTTAAGGGCTGCCCTGCGGACCTAGTGGTGTGTGACGGGGCTCCTGATGTAACCGGTCTCCATGATG TTGATGAGTATATGCAGGCCCAGCTCCTCCTAGCTGCTCTGAACATTGCTACACATGTCCTGAAGCCAGG 20 GGGCTGCTTTGTGGCCAAGATATTCCGAGGCCGGGATGTGACGCTCCTCTACAGCCAGCTGCAGGTCTTC TTCTCCAGCGTGCTGTGTGCCAAGCCCAGGAGCAGCCGGAACTCTAGCATCGAGGCCTTCGCTGTCTGTC AGGGCTATGACCCTCCCGAGGGCTTCATCCCGGACCTGAGCAAACCCCTGCTGGACCATTCTTACGATTT CAACCAGCTGGATGGTCCCACCCGCATCATTGTGCCTTTTGTGACCTGTGGGGACCTGAGCTCCTATGAT TCGGACCGCAGTTACCCACTGGACCTAGAGGGCGGCTCAGAGTACAAGTACACTCCACCCACACAGCCCC 25 CCATCTCGCCACCATACCAGGAGGCCTGCACGTTGAAGAGGAAGGGGCAGCTGGCCAAGGAGATCCGCCC CCAGGACTGCCCCATCAGCAGAGTGGACACGTTTCCCCAGCCCCTGGCCGCCCCTCAGTGCCACACCCTG CTGGCCCCTGAGATGGAAGACAATGAAATGAGTTGTTCACCTTAACCCATTACGCTGGCAAACTGATACA ACTCAGAAACTGCTCAATGTCAGGAAAACCGGAACTTGTTGATGAACTTGTTTTCAAAATATCATCTCAT CAACGAGGCCTGGACAAACAAGCCCTGAGGAGGGATCTGCAACAACCCTGAAGACAACAAGGAAAGAAAC 30 CATGAAAGTCTGTCTGTACTGCAGTGGGAATTCTTGAGTGAGGTCTTACCTCTTCTTTAAACCTCTTCAG GAGTGTCCTGATTATGTCCAGAATTTTCCCTAAAGGCAGGGATTCTTAACCTGGATAGAAGCCAGGGGTA ACCATGAACTTGATGGAAGAAAATGTTACATCTTTATTTTCAGCAATGAAACTGAAATTTAGCCTTACTC CCAAGTTATAAATGCTGGCAACAAATCACAGTAGTAAAAGCAGTACCTGGGATTTTCCCACCAATACAAA CCAGTTACAGCAACTGTAATGTAACATAAAAAGGGCATCTTGAAGTATTGTTCATATTTGTCACCTCTTG 35 GAATGATAGCAGATCCTGTAAGTTGATGTATTAATTAAAAAGCCCATATATTACTGCATCTCAATTTTGT TTCAATTTAATATATAGTAATAATTGTAATTCAATGTAA By “FtsJ RNA 2'-O-methyltransferase 3 polypeptide” or “FTSJ3 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. 26 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 NP_060117.3, or a fragment thereof, and having methyltransferase activity. The sequence of an exemplary FTSJ3 polypeptide is provided below: >NP_060117.3 pre-rRNA 2'-O-ribose RNA methyltransferase FTSJ3 [Homo sapiens] MGKKGKVGKSRRDKFYHLAKETGYRSRSAFKLIQLNRRFQFLQKARALLDLCAAPGGWLQVAAKFMPVSS 5 LIVGVDLVPIKPLPNVVTLQQDITTERCRQALRKELKTWKVDVVLNDGAPNVGASWVHDAYSQAHLTLMA LRLACDFLARGGSFITKVFRSRDYQPLLWIFQQLFRRVQATKPQASRHESAEIFVVCQGFLAPDKVDSKF FDPKFAFKEVEVQAKTVTELVTKKKPKAEGYAEGDLTLYHRTSVTDFLRAANPVDFLSKASEIMVDDEEL AQHPATTEDIRVCCQDIRVLGRKELRSLLNWRTKLRRYVAKKLKEQAKALDISLSSGEEDEGDEEDSTAG TTKQPSKEEEEEEEEEQLNQTLAEMKAQEVAELKRKKKKLLREQRKQRERVELKMDLPGVSIADEGETGM 10 FSLSTIRGHQLLEEVTQGDMSAADTFLSDLPRDDIYVSDVEDDGDDTSLDSDLDPEELAGVRGHQGLRDQ KRMRLTEVQDDKEEEEEENPLLVPLEEKAVLQEEQANLWFSKGSFAGIEDDADEALEISQAQLLFENRRK GRQQQQKQQLPQTPPSCLKTEIMSPLYQDEAPKGTEASSGTEAATGLEGEEKDGISDSDSSTSSEEEESW EPLRGKKRSRGPKSDDDGFEIVPIEDPAKHRILDPEGLALGAVIASSKKAKRDLIDNSFNRYTFNEDEGE LPEWFVQEEKQHRIRQLPVGKKEVEHYRKRWREINARPIKKVAEAKARKKRRMLKRLEQTRKKAEAVVNT 15 VDISEREKVAQLRSLYKKAGLGKEKRHVTYVVAKKGVGRKVRRPAGVRGHFKVVDSRMKKDQRAQQRKEQ KKKHKRK By “FtsJ RNA 2'-O-methyltransferase 3 polynucleotide” or “FTSJ3 polynucleotide” is meant a nucleic acid molecule encoding an FTSJ3 polypeptide or fragment thereof. The sequence of an exemplary FTSJ3 polynucleotide is provided below: 20 >NM_017647.4 Homo sapiens FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), mRNA AGATCCGGTCGCAGGCAGCGTCCAGCATGGACTGGTCCAGGCACCTCCGACCCACCCGACGCTCCTGCCC CGGCCCGCTCTCCACTCAGATCACGCTCGCTCCGCACCCAGCCCCGTAACCTGCCGGGCCGCCACTGCAC CCGCCATACCTGCTCTGGTCCGTCAAGCGCCATCTTCTCTCTTCAGCAGAGACCGCCGGCATCCGAGCCG TCTTGGCGCGCAAGCGCGGAAGCGGAGGATTGGGACCTGAAGACCAGGCAGAGAAGAATTCCGGGTCAAT 25 GGGCGGGGTGTGCGGCGAGACCACACTAAGGGAACCATGATGGGAGGGGCGGCTACTCGAGTTCAGGGGC TTTGGAAGGGGATGAGGAAGGAGTAGAACTCCTCCTCAGTGTTGCCTCATTGAAACTCTGTTTATAGTTC TTGTGATATGCCTTCCTAGTCTCAACTTTCAACTTACAAAACCCAAGGACAGAGTATTGAGAACAATCTA GGGCCACACTCAAAATGGCGGAAAACCTCGAGCCAGGGACGCAGGGGACGCAAGCGGAAGCGGAAGTAGT TAGCTGCATACTTGTGCGGTTCCAAGTGTGGAGAAAGCGGCTCTGGGTCTAGATTGAGGGATACTCCCCC 30 TTTCCACCATGGGCAAGAAGGGCAAAGTTGGCAAGAGCCGACGAGACAAGTTTTATCACTTGGCGAAGGA GACGGGTTACCGTTCCCGATCTGCTTTCAAGCTGATCCAGCTCAATCGCCGCTTTCAGTTCCTGCAGAAA GCCCGAGCCTTGCTGGACCTGTGTGCTGCGCCAGGGGGATGGCTGCAGGTAGCTGCCAAGTTTATGCCTG TATCCAGCCTTATTGTGGGAGTGGACCTGGTTCCAATCAAGCCTCTCCCCAATGTGGTGACTCTCCAGCA GGACATCACAACAGAACGTTGTAGGCAGGCCCTGAGGAAGGAGCTGAAGACCTGGAAGGTTGATGTTGTG 35 CTCAATGATGGGGCCCCCAACGTTGGGGCTAGCTGGGTCCATGATGCTTACTCACAAGCCCATTTGACAC TGATGGCTCTACGTTTGGCTTGTGACTTTTTGGCCCGTGGTGGCAGCTTCATCACAAAGGTTTTCCGTTC TCGTGACTATCAGCCTCTGCTATGGATCTTTCAGCAGCTGTTCCGCCGTGTCCAGGCCACCAAGCCCCAA GCCTCTCGCCATGAATCTGCAGAGATCTTTGTAGTCTGCCAAGGATTCCTGGCCCCTGACAAGGTTGACA GTAAATTCTTTGACCCCAAATTTGCCTTTAAGGAGGTTGAAGTTCAGGCTAAGACCGTTACTGAATTGGT 40 TACTAAGAAGAAGCCAAAGGCTGAAGGCTATGCTGAGGGTGACCTCACTCTCTATCACCGTACCTCAGTC 27 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ACTGACTTCCTCCGAGCTGCCAACCCTGTTGACTTCCTCTCCAAGGCCAGCGAAATCATGGTAGATGATG AAGAGTTGGCACAGCATCCAGCTACCACTGAGGACATACGGGTGTGCTGTCAGGACATCAGAGTGTTGGG GCGCAAGGAGCTCAGGTCGCTACTAAACTGGAGAACAAAACTTCGGCGATATGTGGCCAAGAAGCTGAAA GAACAAGCAAAGGCACTGGACATCAGCCTCAGCTCTGGAGAGGAAGATGAAGGTGATGAGGAGGACTCAA 5 CAGCTGGAACCACAAAGCAGCCCTCTAAGGAGGAGGAGGAAGAGGAGGAGGAGGAACAACTGAACCAGAC CTTGGCAGAAATGAAGGCCCAGGAGGTGGCGGAATTGAAGAGGAAGAAAAAGAAGCTGTTGCGTGAGCAG AGAAAGCAGCGGGAGCGTGTGGAGCTGAAGATGGATCTGCCTGGGGTTTCCATTGCAGACGAGGGGGAGA CTGGCATGTTCTCCTTGAGCACCATCCGGGGTCACCAGTTATTAGAGGAAGTAACACAAGGGGATATGAG TGCAGCAGACACATTTCTGTCCGATCTGCCAAGGGATGATATCTATGTGTCAGATGTTGAGGACGACGGT 10 GATGACACATCTCTGGATAGTGACCTGGATCCAGAGGAGCTGGCAGGAGTCAGGGGACATCAGGGTCTAA GGGACCAAAAGCGTATGCGACTTACTGAAGTGCAAGATGATAAAGAGGAGGAGGAGGAGGAGAATCCACT GCTGGTACCACTGGAGGAAAAGGCAGTACTGCAGGAAGAACAAGCCAACCTGTGGTTCTCAAAGGGCAGC TTTGCTGGGATCGAGGACGATGCCGATGAGGCCCTGGAGATCAGTCAGGCCCAGCTGTTATTTGAGAACC GGCGGAAGGGACGGCAGCAGCAGCAGAAGCAGCAGCTGCCACAGACACCCCCTTCCTGTTTGAAGACTGA 15 GATAATGTCTCCCCTGTACCAAGATGAAGCCCCTAAGGGAACAGAGGCTTCTTCGGGGACAGAAGCTGCC ACTGGCCTTGAAGGGGAAGAAAAGGATGGCATCTCAGACAGTGATAGCAGTACTAGCAGTGAGGAAGAAG AGAGCTGGGAACCCCTCCGTGGTAAGAAGCGAAGCCGTGGGCCTAAGTCAGATGATGACGGGTTTGAGAT AGTGCCTATTGAGGACCCAGCGAAACATCGGATACTGGACCCCGAAGGCCTTGCTCTAGGTGCTGTTATT GCCTCTTCCAAAAAGGCCAAGAGAGACCTCATAGATAACTCCTTCAACCGGTACACATTTAATGAGGATG 20 AGGGGGAGCTTCCGGAGTGGTTTGTGCAAGAGGAAAAGCAGCACCGGATACGACAGTTGCCTGTTGGTAA GAAGGAGGTGGAGCATTACCGGAAACGCTGGCGGGAAATCAATGCACGTCCCATCAAGAAGGTGGCTGAG GCTAAGGCTAGAAAGAAAAGGAGGATGCTGAAGAGGCTGGAGCAGACCAGGAAGAAGGCAGAAGCCGTGG TGAACACAGTGGACATCTCAGAACGAGAGAAAGTGGCACAGCTGCGAAGTCTCTACAAGAAGGCTGGGCT TGGCAAGGAGAAACGCCATGTCACCTACGTTGTAGCCAAAAAAGGTGTGGGCCGCAAAGTGCGCCGGCCA 25 GCTGGAGTCAGAGGTCATTTCAAGGTGGTGGACTCAAGGATGAAGAAGGACCAAAGAGCACAGCAACGTA AGGAACAAAAGAAAAAACACAAACGGAAGTAAGCAGAGCTGCCAGGCTCCCAGGAGAGCATGGGGACTAG GAGGAAGGGTGTGGCATGGCTCAGTCTGGCCCCCTTGATTACCGGCCTAGCCCCTGCTCACATCACAGCT GTCTGAAGAACAGTGAGGTGGAGTGCCTAGAACTCCCGTGGTGGTCCTGAGCAGAGAGGAGGATGTCCTC CTGCCTGCCTGAAGGTCTCCCATGAAAACACTGCTGAACTGTGTTGACACTCATGACCCTTTTTTTAAAC 30 CGTTAAAGGGAAGTTCGGTGTTGGAGCGATACTCAATGTAGTCAGTCTACACCTGGACGTGTGGGCCACT TAAGCCCTCCCCACCCCCATCCTATTCCTGAATAAAACCAGGATAATGGAA By “HEN methyltransferase 1 polypeptide” or “HENMT1 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001096062.1 or NP_653185.2, or a fragment thereof, and having methyltransferase activity. 35 The sequences of exemplary HENMT1 polypeptides are provided below: >NP_001096062.1 small RNA 2'-O-methyltransferase [Homo sapiens] MEENNLQCSSVVDGNFEEVPRETAIQFKPPLYRQRYQFVKNLVDQHEPKKVADLGCGDTSLLRLLKVNPC IELLVGVDINEDKLRWRGDSLAPFLGDFLKPRDLNLTITLYHGSVVERDSRLLGFDLITCIELIEHLDSG DLARFPEVVFGYLSPSMIVISTPNSEFNPLFPSVTLRDSDHKFEWTRMEFQTWALYVANRYDYSVEFTGV 40 GEPPAGAENVGYCTQIGIFRKNGGKATESCLSEQHDQHVYKAVFTTSYPSLQQERFFKLVLVNEVSQQVE 28 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 SLRVSHLPRRKEQAGERGDKPKDIGGSKAPVPCFGPVFTEVEKAKIENSPTPFCVGDKFFVPLQRLLAYP KLNRLCANEEMMRSVIADSIPLSSDGSAVVADLRNYFDEQFEF >NP_653185.2 small RNA 2'-O-methyltransferase [Homo sapiens] 5 MEENNLQCSSVVDGNFEEVPRETAIQFKPPLYRQRYQFVKNLVDQHEPKKVADLGCGDTSLLRLLKVNPC IELLVGVDINEDKLRWRGDSLAPFLGDFLKPRDLNLTITLYHGSVVERDSRLLGFDLITCIELIEHLDSG DLARFPEVVFGYLSPSMIVISTPNSEFNPLFPSVTLRDSDHKFEWTRMEFQTWALYVANRYDYSVEFTGV GEPPAGAENVGYCTQIGIFRKNGGKATESCLSEQHDQHVYKAVFTTSYPSLQQERFFKLVLVNEVSQQVE SLRVSHLPRRKEQAGERGDKPKDIGGSKAPVPCFGPVFTEVEKAKIENSPTPFCVGDKFFVPLQRLLAYP 10 KLNRLCANEEMMRSVIADSIPLSSDGSAVVADLRNYFDEQFEF By “HEN methyltransferase 1 polynucleotide” or “HENMT1 polynucleotide” is meant a nucleic acid molecule encoding an HENMT1 polypeptide or fragment thereof. The sequences of exemplary HENMT1 polynucleotides are provided below: 15 >NM_001102592.2 Homo sapiens HEN methyltransferase 1 (HENMT1), transcript variant 2, mRNA GGGCGTCAGGGCGGCGCTGAGTTGAGGACGCTGGCGGCTCCGCGACGAGCTTTGTTTTTTCGGTTGGGCA TGCTGCTTACCGCAGGATGATGGAAGCGACGGCCCGTACAAAGACAGAAGCGTTGGTTTCACTGAGTCAG TGCTGATTCTCTCAAATAGAGCTTGAAGGATAAATCTTCATTTTTGTTTCAACAAAACTTCGAAACAAAA 20 TGGAAGAAAATAATCTACAGTGCAGTAGTGTGGTTGACGGTAATTTTGAAGAAGTTCCCAGGGAGACGGC AATTCAGTTTAAACCTCCACTATACAGACAGCGGTACCAGTTCGTTAAAAATTTAGTGGATCAACATGAG CCTAAGAAGGTTGCAGACCTGGGATGTGGTGATACTTCACTCTTAAGGCTGCTAAAAGTCAATCCATGCA TTGAATTGCTTGTTGGAGTAGATATTAATGAGGATAAATTACGATGGAGAGGGGATTCGTTAGCTCCTTT CCTGGGGGATTTTCTGAAACCTCGGGATCTGAATTTGACCATCACATTGTATCATGGCTCCGTTGTGGAG 25 AGAGACTCTCGTTTGCTTGGATTTGACTTGATAACGTGTATTGAATTAATAGAACATTTGGATTCAGGTG ATCTGGCCAGATTTCCTGAAGTGGTATTTGGGTACCTGTCTCCATCCATGATTGTCATCAGCACACCAAA CTCTGAATTCAATCCCCTGTTTCCATCAGTGACCTTAAGAGATTCAGATCATAAATTTGAGTGGACCAGA ATGGAGTTTCAGACCTGGGCTTTATATGTGGCAAATCGCTATGATTACTCTGTGGAGTTTACTGGTGTCG GGGAACCACCAGCTGGAGCTGAGAATGTTGGATACTGTACCCAGATAGGAATCTTCCGGAAAAATGGAGG 30 AAAGGCAACAGAATCATGTCTTTCAGAGCAGCATGATCAGCATGTTTATAAAGCTGTTTTTACCACCTCA TACCCAAGCTTACAGCAGGAAAGGTTCTTTAAACTTGTGTTGGTTAATGAGGTGTCCCAACAAGTGGAAA GCTTAAGAGTGAGCCACCTGCCAAGGCGGAAAGAACAGGCTGGGGAACGGGGTGATAAGCCCAAAGACAT TGGTGGCTCAAAGGCCCCTGTCCCATGCTTTGGACCAGTCTTCACAGAGGTTGAGAAGGCCAAGATAGAG AACTCTCCCACACCCTTCTGTGTTGGAGATAAATTTTTCGTACCTCTGCAGAGACTCCTTGCGTATCCCA 35 AGTTGAACCGCTTATGTGCTAATGAAGAGATGATGAGATCAGTCATTGCTGACTCAATTCCTCTGAGCAG TGATGGTTCTGCAGTGGTGGCTGACCTGCGTAATTATTTTGATGAACAGTTTGAGTTTTGAACCATGTTT ATTTCCTGAAATTTCAGGGTCTCAGCGATAGTTGTGCTCACTTAGAATTTAGTTTTTTTTGTGTAATCCT AATTCAAGTAATGTTTTTAAAGTTTCACTGCAAAAGTCTATGTTCCAAGCCATTGGACAGACCTGCTTGA GATATGGCCAGACTGCAGTGAGCCCTGAGAAAGATATGAGGGTTTAAAACGGGTGCTTTCCTTTGATTTT 40 GGACTTTTTTGTTTTCTCAAGAATAAAGAAGTTGGATGTGGTAATATGTTAA 29 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_144584.3 Homo sapiens HEN methyltransferase 1 (HENMT1), transcript variant 1, mRNA AAGCCGCCTTCCGGCTCGCGGTCTGCGGACGCGCTCGGAGTTGGGGGCCTTCGCCAACAGCTGCCGTACC 5 GTGCCGCGTCGCAGCCGCACGCTAGCGGGGTCAACGGCGCGGCAGGCGCAGGGGTCCTCGCCAAGGTTCG GAAGAGGACCTTGAAAATGGCTTCTGGGCATGCGCCACAGCGGCCGTTACGAGACGCAAAACCGACGCCT TGGAACTGAGAGCCTAGAGGGACGGAGCTCGGCGCACCATGAGGCTCCCGGGGCCCAAGGCCGACCGTGA GAGCGCACGAGCTGAGTCAGTGCTGATTCTCTCAAATAGAGCTTGAAGGATAAATCTTCATTTTTGTTTC AACAAAACTTCGAAACAAAATGGAAGAAAATAATCTACAGTGCAGTAGTGTGGTTGACGGTAATTTTGAA 10 GAAGTTCCCAGGGAGACGGCAATTCAGTTTAAACCTCCACTATACAGACAGCGGTACCAGTTCGTTAAAA ATTTAGTGGATCAACATGAGCCTAAGAAGGTTGCAGACCTGGGATGTGGTGATACTTCACTCTTAAGGCT GCTAAAAGTCAATCCATGCATTGAATTGCTTGTTGGAGTAGATATTAATGAGGATAAATTACGATGGAGA GGGGATTCGTTAGCTCCTTTCCTGGGGGATTTTCTGAAACCTCGGGATCTGAATTTGACCATCACATTGT ATCATGGCTCCGTTGTGGAGAGAGACTCTCGTTTGCTTGGATTTGACTTGATAACGTGTATTGAATTAAT 15 AGAACATTTGGATTCAGGTGATCTGGCCAGATTTCCTGAAGTGGTATTTGGGTACCTGTCTCCATCCATG ATTGTCATCAGCACACCAAACTCTGAATTCAATCCCCTGTTTCCATCAGTGACCTTAAGAGATTCAGATC ATAAATTTGAGTGGACCAGAATGGAGTTTCAGACCTGGGCTTTATATGTGGCAAATCGCTATGATTACTC TGTGGAGTTTACTGGTGTCGGGGAACCACCAGCTGGAGCTGAGAATGTTGGATACTGTACCCAGATAGGA ATCTTCCGGAAAAATGGAGGAAAGGCAACAGAATCATGTCTTTCAGAGCAGCATGATCAGCATGTTTATA 20 AAGCTGTTTTTACCACCTCATACCCAAGCTTACAGCAGGAAAGGTTCTTTAAACTTGTGTTGGTTAATGA GGTGTCCCAACAAGTGGAAAGCTTAAGAGTGAGCCACCTGCCAAGGCGGAAAGAACAGGCTGGGGAACGG GGTGATAAGCCCAAAGACATTGGTGGCTCAAAGGCCCCTGTCCCATGCTTTGGACCAGTCTTCACAGAGG TTGAGAAGGCCAAGATAGAGAACTCTCCCACACCCTTCTGTGTTGGAGATAAATTTTTCGTACCTCTGCA GAGACTCCTTGCGTATCCCAAGTTGAACCGCTTATGTGCTAATGAAGAGATGATGAGATCAGTCATTGCT 25 GACTCAATTCCTCTGAGCAGTGATGGTTCTGCAGTGGTGGCTGACCTGCGTAATTATTTTGATGAACAGT TTGAGTTTTGAACCATGTTTATTTCCTGAAATTTCAGGGTCTCAGCGATAGTTGTGCTCACTTAGAATTT AGTTTTTTTTGTGTAATCCTAATTCAAGTAATGTTTTTAAAGTTTCACTGCAAAAGTCTATGTTCCAAGC CATTGGACAGACCTGCTTGAGATATGGCCAGACTGCAGTGAGCCCTGAGAAAGATATGAGGGTTTAAAAC GGGTGCTTTCCTTTGATTTTGGACTTTTTTGTTTTCTCAAGAATAAAGAAGTTGGATGTGGTAATATGTT 30 AA As used herein, a(n) "heterologous" or "exogenous" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule, or portion of a or nucleic acid molecule that is not native to a host cell but can be homologous to a nucleic acid molecule or portion thereof from 35 the host cell. The source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule (i.e., not endogenous or native) is added to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host genome or exist as extra- 30 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector) and can be present in multiple copies. In addition, "heterologous" may also refer to a non-native enzyme, protein, polypeptide, or other activity, (e.g., directly introduced or encoded by an exogenous nucleic acid molecule) introduced into the host cell, even if the host cell encodes a homologous 5 protein or activity. Moreover, a cell comprising a "modification” or a "heterologous" polynucleotide or binding protein includes progeny of that cell, regardless of whether the progeny were themselves transduced, transfected, or otherwise manipulated or changed. A “host cell” or “cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that 10 have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell. "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of 15 hydrogen bonds. The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is 20 sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using 25 analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. 30 By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate 31 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. 5 By an "isolated polypeptide" is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In an embodiment, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the disclosure. An isolated 10 polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. By “mitochondrial rRNA methyltransferase 1 polypeptide” or “MRM1 polypeptide” is 15 meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_079140.2, or a fragment thereof, and having methyltransferase activity. The sequence of an exemplary MRM1 polypeptide is provided below: >NP_079140.2 rRNA methyltransferase 1, mitochondrial precursor [Homo sapiens] MALLSTVRGATWGRLVTRHFSHAARHGERPGGEELSRLLLDDLVPTSRLELLFGMTPCLLALQAARRSVA 20 RLLLQAGKAGLQGKRAELLRMAEARDIPVLRPRRQKLDTMCRYQVHQGVCMEVSPLRPRPWREAGEASPG DDPQQLWLVLDGIQDPRNFGAVLRSAHFLGVDKVITSRRNSCPLTPVVSKSSAGAMEVMDVFSTDDLTGF LQTKAQQGWLVAGTVGCPSTEDPQSSEIPIMSCLEFLWERPTLLVLGNEGSGLSQEVQASCQLLLTILPR RQLPPGLESLNVSVAAGILLHSICSQRKGFPTEGERRQLLQDPQEPSARSEGLSMAQHPGLSSGPEKERQ NEG 25 By “mitochondrial rRNA methyltransferase 1 polynucleotide” or “MRM1 polynucleotide” is meant a nucleic acid molecule encoding an MRM1 polypeptide or fragment thereof. The sequence of an exemplary MRM1 polynucleotide is provided below: >NM_024864.5 Homo sapiens mitochondrial rRNA methyltransferase 1 (MRM1), 30 mRNA; nuclear gene for mitochondrial product GAACCCGGAAGCGAGGGACCCACGTGGGAGCCTGGGAGCGGGTGGTCGTAGCTCGGTAGTCCAGTTGTGG GTAATCGGGGCTGTTTGTTCCTGTCCGAGAGAGCTCGGCGGAGACGGCTGTCGAGTACCCTTCACCTCGG TGTTGGGAGCCTGGGAGCGAACTGCGGCGCGGGTTACCGCTCCCGGGGACGCAGCAAGGGGCATCGAGTC CCTGGCGGGAGCTGCGCCATGGCATTGCTCTCGACCGTCCGGGGCGCGACCTGGGGTCGCCTCGTCACCC 35 GTCATTTCTCCCATGCAGCGCGGCATGGGGAGCGGCCTGGTGGGGAGGAGCTAAGCCGCTTGCTGCTGGA TGACCTGGTGCCGACCTCTCGGCTGGAGCTTCTGTTTGGCATGACCCCGTGTCTCCTGGCTCTGCAGGCC GCCCGCCGCTCTGTGGCCCGGCTCCTGCTCCAGGCGGGTAAAGCTGGGCTGCAGGGGAAGCGGGCCGAGC 32 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGCTCCGGATGGCCGAGGCGCGGGACATTCCAGTTCTGCGGCCCAGACGGCAGAAACTGGACACAATGTG CCGCTACCAGGTCCACCAGGGTGTCTGCATGGAGGTGAGCCCGCTGCGGCCCCGGCCTTGGAGAGAGGCC GGGGAGGCGAGCCCAGGCGACGACCCCCAGCAGTTGTGGCTCGTCCTCGATGGGATCCAGGATCCCCGGA ATTTTGGGGCTGTGCTGCGTTCCGCACACTTCCTCGGAGTGGATAAGGTCATCACCAGCCGGAGAAACAG 5 CTGCCCGCTCACTCCAGTAGTCAGCAAGTCCAGCGCGGGGGCTATGGAGGTGATGGACGTGTTCTCCACT GATGACCTCACCGGATTTTTACAGACCAAAGCCCAGCAGGGCTGGCTCGTGGCCGGCACGGTGGGCTGCC CAAGCACAGAGGATCCCCAGTCCTCCGAGATCCCCATCATGAGTTGCTTGGAGTTCCTCTGGGAACGGCC TACTCTCCTTGTGCTGGGGAATGAGGGCTCAGGTCTATCCCAGGAGGTGCAGGCCTCCTGCCAGCTTCTC CTCACCATCCTGCCCCGGCGCCAGCTGCCTCCTGGACTTGAGTCCTTGAACGTCTCTGTGGCTGCAGGAA 10 TTCTTCTTCACTCCATTTGCAGCCAGAGGAAGGGTTTCCCCACAGAGGGGGAGAGAAGGCAGCTTCTCCA AGACCCCCAAGAACCCTCAGCCAGGTCTGAAGGGCTCAGCATGGCTCAGCACCCAGGGCTGTCTTCAGGC CCAGAGAAAGAGAGGCAAAATGAGGGCTGACGTGGACTGTCCACAGTGTTCATGTGCTGGAGTCAGGGAC GGCCGCACCTGCCTCCGCCGGCTCCAGTGTGCGGGGAGCCTCTGCCTGAGTGTGCACCAGGCCCATGTTT ATTGACCACAGTCTGGGGGGGGGGGAAGGGGACTGCGGTGGACACCAGAGGAAGCTGTTTCCTGTTGTGA 15 TGTTGGACCTGTAGTAGGACATGGTGATTTGTTAATTTCCATGGGAAGCCATGATGGCCTAGCATGGAGG GAATCTGTTCCCAGGCCCTGCCTGGAAGTTGAGGGAAAGTTTAGACATCTGCAGAGAGGCAGGCAGCCCA GCCCAGGGGACCCGTTCCTCTTGAACCAGTCATTGCCTGTGGCAAATGTGTGTATGAGAATGTGGGGGGT GGAGGGCGGGGCCCTGATGTGGAGTAGACAGTGCGCACCTCAGGCCCACACACGGCCCCGCCCTGGGGCC TTGAGCGCAGGCCTCATCTTTCTGTGCCGCGGGACTCCGCACCTACCTCACAGGGTTGTTGTGAGGCTCA 20 AATAAAACATCACTCAGCA By “mitochondrial rRNA methyltransferase 2 polypeptide” or “MRM2 polypeptide” or “FTSJ2 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_037525.1, or a fragment thereof, and having 25 methyltransferase activity. The sequence of an exemplary MRM2 polypeptide is provided below: >NP_037525.1 rRNA methyltransferase 2, mitochondrial [Homo sapiens] MAGYLKLVCVSFQRQGFHTVGSRCKNRTGAEHLWLTRHLRDPFVKAAKVESYRCRSAFKLLEVNERHQIL RPGLRVLDCGAAPGAWSQVAVQKVNAAGTDPSSPVGFVLGVDLLHIFPLEGATFLCPADVTDPRTSQRIL 30 EVLPGRRADVILSDMAPNATGFRDLDHDRLISLCLTLLSVTPDILQPGGTFLCKTWAGSQSRRLQRRLTE EFQNVRIIKPEASRKESSEVYFLATQYHGRKGTVKQ By “mitochondrial rRNA methyltransferase 2 polynucleotide” or “MRM2 polynucleotide” or “FTSJ2 polynucleotide” is meant a nucleic acid molecule encoding an 35 MRM1 polypeptide or fragment thereof. The sequence of an exemplary MRM2 polynucleotide is provided below: >NM_013393.3 Homo sapiens mitochondrial rRNA methyltransferase 2 (MRM2), mRNA; nuclear gene for mitochondrial product ACTTCCGGCGGCGCGCTGCAGGCGCGGGGAACACCAATGGCGGGGTACTTGAAGCTGGTGTGTGTTTCCT 33 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TTCAGCGTCAAGGGTTCCACACTGTTGGGAGTCGCTGCAAGAATCGGACAGGCGCTGAGCACCTGTGGCT GACCCGACATCTCAGGGACCCATTTGTGAAGGCTGCGAAGGTGGAGAGTTACCGGTGTCGAAGCGCCTTC AAGCTCCTGGAGGTGAACGAGAGGCACCAGATTCTGCGGCCCGGCCTTCGGGTGTTAGACTGTGGGGCAG CTCCTGGGGCCTGGAGTCAGGTGGCGGTGCAGAAGGTCAACGCCGCAGGCACAGATCCCAGCTCTCCTGT 5 TGGCTTCGTGCTTGGGGTAGATCTTCTTCACATATTCCCCCTGGAAGGAGCAACTTTTCTGTGCCCTGCT GACGTGACTGACCCGAGAACCTCACAGAGAATCCTCGAGGTGCTTCCTGGCAGGAGAGCAGATGTGATTC TGAGCGACATGGCGCCCAATGCCACAGGGTTCCGGGACCTCGATCATGACAGGCTCATCAGCCTGTGCCT GACCCTTCTCAGCGTGACCCCAGACATCCTGCAACCTGGGGGGACATTCCTTTGTAAAACCTGGGCTGGA AGTCAAAGCCGTCGGTTACAGAGGAGACTGACAGAGGAATTCCAGAATGTAAGGATCATCAAACCTGAAG 10 CCAGCAGGAAAGAGTCATCAGAAGTGTACTTCTTGGCCACACAGTACCACGGAAGGAAGGGCACTGTGAA GCAGTGAGGATTTCTTGTGCCATTTTCATAATGGTCATTAGCTCCTTTTAAGCTAGAAACGTAGCCTGAG CTCCTGAAGAGTTCCTGGGAGATTTGAGCTGATTTTGGAGATGGAGCAGGACAAGTGGGGAGTCTCTCTC TCTCTTTCTCTCTCTCTCTTTTTAACCAAAAAGAGATGACAAAACTAAGTTCAGGGGCCATGGAAAATGA AAAAGTCCGCTATATTGTGATTTGGGAAGAGAAAGTTATCAAGAGAAAGAGGTGAGGATGGAAGGATGGA 15 GAAAAACAGACTGTGGGAAGGATCAGAAGGAATCCGCCGAGGCAGGGATGGGTGTGCCCATGTGTGCCTT GACGGGACTTCATCTTATAGACTGTTAAACTGTCACACACAAACAGGCTTTCCACCCCTGCTCTGAGAGC ACCACGCACAGATTTCCAGTTCTTAGTGTGGCTGTTTAAAGTAGAAAATCTGGGGGCTGGGTGAGGCCAC TCATGCCTGTAAACCCAGGGCTTTAGAAGGCTGAGGCTGGGGGATTGCTTGAAGTCAGGAGTTCAAGACC AACCTGGGCAACATAGCAACACCCCCCATGTCTACAAAAATGAAAAACCAAAAAGCAAACCAAAAGAAAA 20 ATCTGAAATTTCCATCTGGGGATTAACTTCTGTCTTTCTGGTGAACAATATAGCAATTCACGCATTCTTC AAGCAGCAAAAGTTCCCGGAACAATTAGGGAAGACGTATGGTCTGAATTTATCCAGGCAGTGGGTCTGCT TTGGTTTTTGCTGGAAATTTATATCAGTGTCTGGGCTCCCAAGAACATAAATGTAATTGCCAAAGCAAGC AGTGATGTGGTGTGTTTATTTTCTTTTACTCATCTAGGAACTTGACGCAGCTTTATATTTAATAAAATAA TAGAGATTGCATTATTTTAAGGTA 25 By “mitochondrial rRNA methyltransferase 3 polypeptide” or “MRM3 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001304876.1 or NP_060616.1, or a fragment thereof, and having methyltransferase activity. The sequences of exemplary MRM3 polypeptides are provided 30 below: >NP_001304876.1 rRNA methyltransferase 3, mitochondrial isoform 2 [Homo sapiens] MTYPKTQLQHSLPLLLICDNLRDPGNLGTILRSAAGAGCSKVLLTKGCVDAWEPKVLRAGMGAHFRMPII NNLEWETVPNYLPPDTRVYVADNCGLYAQAEMSNKASDHGWVCDQRVMKFHKYEEEEDVETGASQDWLPH 35 VEVQSYDSDWTEAPAAVVIGGETYGVSLESLQLAESTGGKRLLIPVVPGVDSLNSAMAASILLFEGKRQL RGRAEDLSRDRSYH >NP_060616.1 rRNA methyltransferase 3, mitochondrial isoform 1 precursor [Homo sapiens] 40 MAALVRPARFVVRPLLQVVQAWDLDARRWVRALRRSPVKVVFPSGEVVEQKRAPGKQPRKAPSEASAQEQ 34 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 REKQPLEESASRAPSTWEESGLRYDKAYPGDRRLSSVMTIVKSRPFREKQGKILLEGRRLISDALKAGAV PKMFFFSRLEYLKELPVDKLKGVSLIKVKFEDIKDWSDLVTPQGIMGIFAKPDHVKMTYPKTQLQHSLPL LLICDNLRDPGNLGTILRSAAGAGCSKVLLTKGCVDAWEPKVLRAGMGAHFRMPIINNLEWETVPNYLPP DTRVYVADNCGLYAQAEMSNKASDHGWVCDQRVMKFHKYEEEEDVETGASQDWLPHVEVQSYDSDWTEAP 5 AAVVIGGETYGVSLESLQLAESTGGKRLLIPVVPGVDSLNSAMAASILLFEGKRQLRGRAEDLSRDRSYH By “mitochondrial rRNA methyltransferase 3 polynucleotide” or “MRM3 polynucleotide” is meant a nucleic acid molecule encoding an MRM3 polypeptide or fragment thereof. The sequences of exemplary MRM3 polynucleotides are provided below: 10 >NM_001317947.2 Homo sapiens mitochondrial rRNA methyltransferase 3 (MRM3), transcript variant 2, mRNA; nuclear gene for mitochondrial product GTGACGCAGCCCGGGTCTCAGGGAACATGGCGGCGCTGGTGAGACCCGCGAGGTTTGTCGTGCGACCGTT GCTGCAGGTGGTCCAGGCTTGGGACCTTGACGCGAGGCGCTGGGTCCGGGCGCTGCGGCGGAGCCCAGTG AAAGTGGTGTTTCCTTCCGGAGAGGTGGTGGAACAGAAGCGCGCTCCTGGGAAGCAGCCCCGCAAGGCAC 15 CATCTGAGGCCAGTGCCCAGGAGCAACGAGAGAAACAACCGCTCGAGGAGTCCGCATCCCGCGCTCCCAG CACCTGGGAAGAGTCTGGGCTTCGCTACGATAAAGCTTATCCCGGGGACAGGAGGCTGAGGGATTTTTGC CAAGCCTGACCATGTTAAGATGACATATCCAAAGACTCAGCTTCAGCATTCACTGCCTTTATTATTGATT TGTGACAATCTCCGTGACCCTGGGAACCTGGGGACAATTCTGAGATCTGCAGCTGGGGCAGGCTGCAGCA AAGTGTTACTCACCAAAGGCTGTGTGGATGCCTGGGAGCCCAAAGTGCTCCGGGCGGGTATGGGCGCACA 20 TTTCCGGATGCCCATTATCAATAATCTGGAATGGGAAACCGTGCCCAATTACCTGCCCCCTGACACTCGG GTCTATGTGGCTGACAACTGTGGCCTTTATGCCCAGGCTGAGATGTCTAATAAAGCTAGTGACCATGGCT GGGTGTGTGATCAACGAGTGATGAAGTTTCACAAGTATGAGGAAGAGGAAGATGTAGAAACCGGAGCCAG TCAAGATTGGCTGCCTCATGTTGAGGTTCAGAGTTACGACTCGGACTGGACAGAGGCGCCGGCAGCTGTG GTGATTGGCGGGGAGACCTACGGCGTGAGCCTGGAGTCCCTGCAGCTGGCCGAGAGCACTGGTGGCAAGA 25 GGCTGCTGATCCCCGTTGTGCCTGGTGTGGACAGCCTCAACTCGGCCATGGCGGCAAGCATCCTGCTTTT CGAAGGGAAAAGACAGCTGCGGGGGAGGGCGGAGGACTTGAGCAGGGACAGGAGTTACCACTGAGGACGC AGAAGTGACTTCTGCTTGAGGACGTCTGCAGCTCCTCCTACACCAGCACACTGGTGGGAGGCTGGCGGAG TCAGTGACTATGGCCCCCACGTTCAGGAGGAAGGTGTGATGCCGTCATACAGTTACAGGAAAAATAAGAA CTTCCTCAGAAAGAACAGGTCCGAATTCTTCCTGTCGCGTCACTGATTTTGAGGTTCTTTTTTCTCTTGG 30 TGACAATAGGTGACCCACGTGGCTCTGTGTGTTTTTAAAAATTGTCCACCAAGAAGCACTTTGTGCCCAG AAAGTTCCTGAAGCATCATCCTGGCAGGGAGGCGCCTGCTCCACCAGCTGGTGGGTGTTTGTAATCGCCA AGCACCAGCTATAGGTCACAGCCACATCACTCACAGCTGATCACTGGTTGGTGGAAAATAAACTATGAGC AGCAGATTACGTTA 35 >NM_018146.4 Homo sapiens mitochondrial rRNA methyltransferase 3 (MRM3), transcript variant 1, mRNA; nuclear gene for mitochondrial product GTGACGCAGCCCGGGTCTCAGGGAACATGGCGGCGCTGGTGAGACCCGCGAGGTTTGTCGTGCGACCGTT GCTGCAGGTGGTCCAGGCTTGGGACCTTGACGCGAGGCGCTGGGTCCGGGCGCTGCGGCGGAGCCCAGTG AAAGTGGTGTTTCCTTCCGGAGAGGTGGTGGAACAGAAGCGCGCTCCTGGGAAGCAGCCCCGCAAGGCAC 40 CATCTGAGGCCAGTGCCCAGGAGCAACGAGAGAAACAACCGCTCGAGGAGTCCGCATCCCGCGCTCCCAG 35 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CACCTGGGAAGAGTCTGGGCTTCGCTACGATAAAGCTTATCCCGGGGACAGGAGGCTGAGCAGTGTAATG ACAATAGTAAAGTCCAGGCCATTTCGGGAAAAACAAGGGAAGATCCTGCTGGAAGGTCGCAGGCTCATTT CAGACGCTCTCAAGGCTGGAGCTGTGCCAAAAATGTTCTTCTTTAGCCGTCTAGAATACCTAAAGGAGTT GCCAGTCGATAAGCTGAAAGGTGTCAGCCTCATTAAGGTGAAATTTGAGGATATCAAGGATTGGTCCGAC 5 CTCGTAACGCCACAAGGAATAATGGGGATTTTTGCCAAGCCTGACCATGTTAAGATGACATATCCAAAGA CTCAGCTTCAGCATTCACTGCCTTTATTATTGATTTGTGACAATCTCCGTGACCCTGGGAACCTGGGGAC AATTCTGAGATCTGCAGCTGGGGCAGGCTGCAGCAAAGTGTTACTCACCAAAGGCTGTGTGGATGCCTGG GAGCCCAAAGTGCTCCGGGCGGGTATGGGCGCACATTTCCGGATGCCCATTATCAATAATCTGGAATGGG AAACCGTGCCCAATTACCTGCCCCCTGACACTCGGGTCTATGTGGCTGACAACTGTGGCCTTTATGCCCA 10 GGCTGAGATGTCTAATAAAGCTAGTGACCATGGCTGGGTGTGTGATCAACGAGTGATGAAGTTTCACAAG TATGAGGAAGAGGAAGATGTAGAAACCGGAGCCAGTCAAGATTGGCTGCCTCATGTTGAGGTTCAGAGTT ACGACTCGGACTGGACAGAGGCGCCGGCAGCTGTGGTGATTGGCGGGGAGACCTACGGCGTGAGCCTGGA GTCCCTGCAGCTGGCCGAGAGCACTGGTGGCAAGAGGCTGCTGATCCCCGTTGTGCCTGGTGTGGACAGC CTCAACTCGGCCATGGCGGCAAGCATCCTGCTTTTCGAAGGGAAAAGACAGCTGCGGGGGAGGGCGGAGG 15 ACTTGAGCAGGGACAGGAGTTACCACTGAGGACGCAGAAGTGACTTCTGCTTGAGGACGTCTGCAGCTCC TCCTACACCAGCACACTGGTGGGAGGCTGGCGGAGTCAGTGACTATGGCCCCCACGTTCAGGAGGAAGGT GTGATGCCGTCATACAGTTACAGGAAAAATAAGAACTTCCTCAGAAAGAACAGGTCCGAATTCTTCCTGT CGCGTCACTGATTTTGAGGTTCTTTTTTCTCTTGGTGACAATAGGTGACCCACGTGGCTCTGTGTGTTTT TAAAAATTGTCCACCAAGAAGCACTTTGTGCCCAGAAAGTTCCTGAAGCATCATCCTGGCAGGGAGGCGC 20 CTGCTCCACCAGCTGGTGGGTGTTTGTAATCGCCAAGCACCAGCTATAGGTCACAGCCACATCACTCACA GCTGATCACTGGTTGGTGGAAAATAAACTATGAGCAGCAGATTACGTTA By “modified nucleobase” or “modified polynucleotide” is meant a polynucleotide comprising one or more alterations that do not occur in a reference polynucleotide, that occur at 25 reduced levels in a reference nucleobase or polynucleotide, or that occur at different positions in the reference polynucleotide. In one embodiment, the modified polynucleotide is an RNA (e.g., mRNA) or a DNA/RNA hybrid. In another embodiment, the mRNA is chemically synthesized or in vitro transcribed. In another embodiment, the modified mRNA comprises one or more internal nucleobases comprising a 2’-O-methyl modification. In another embodiment, the 30 modified mRNA comprises a modification at a position between the 5’ and 3’ termini (i.e., a modification at an internal position) and shows increased stability relative to a reference polynucleotide. By “operably linked” refers to a functional linkage between a regulatory sequence and a coding sequence, where a first polynucleotide is positioned adjacent to a second polynucleotide 35 that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide. The described components are therefore in a relationship permitting them to function in their intended manner. For example, placing a coding sequence under regulatory control of a promoter means 36 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 positioning the coding sequence such that the expression of the coding sequence is controlled by the promoter. By “portion” is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the 5 reference nucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides. By “positioned for expression” is meant that the polynucleotide of the disclosure (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a polynucleotide or 10 polypeptide described herein). The term “promoter” as used herein refers to a sequence of DNA that directs the expression (transcription) of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene. A promoter may be “inducible”, initiating transcription in response to an inducing agent or, in contrast, a promoter may be “constitutive”, whereby an inducing agent does15 not regulate the rate of transcription. A promoter may be regulated in a tissue-specific or tissue- preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types. As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. 20 By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. By “reference” is meant a standard or control condition. In embodiments, a reference polynucleotide (e.g., mRNA) is an unmodified or wild type version of a polynucleotide (e.g., mRNA) having the same or substantially the same sequence as a modified mRNA. In some embodiments, the reference polynucleotide (e.g., mRNA) is a chemically synthesized or in vitro 25 transcribed mRNA. A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 30 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, or even about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. 37 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an 5 endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial 10 identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol.152:399; Kimmel, A. R. (1987) Methods Enzymol. 15 152:507). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, and less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency 20 hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, or at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels 25 of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM 30 trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing 38 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C. In an embodiment, 5 wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In an embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In an embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and 10 are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. 15 By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In an embodiment, such a sequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for 20 comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar 25 sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a 30 probability score between e-3 and e-100 indicating a closely related sequence. By “27S pre-rRNA (guanosine2922-2'-O)-methyltransferase polypeptide” or “SPB1 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_009877.1, or a fragment thereof, and having methyltransferase activity. The sequence of an exemplary SPB1 polypeptide is provided below: 39 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_009877.127S pre-rRNA (guanosine2922-2'-O)-methyltransferase [Saccharomyces cerevisiae S288C] MGKTQKKNSKGRLDRYYYLAKEKGYRARSSFKIIQINEKYGHFLEKSKVVIDLCAAPGSWCQVASKLCPV NSLIIGVDIVPMKPMPNVITFQSDITTEDCRSKLRGYMKTWKADTVLHDGAPNVGLGWVQDAFTQSQLTL 5 QALKLAVENLVVNGTFVTKIFRSKDYNKLIWVFQQLFEKVEATKPPASRNVSAEIFVVCKGFKAPKRLDP RLLDPKEVFEELPDGQQNMESKIYNPEKKVRKRQGYEEGDNLLYHETSILDFVRTEDPISMLGEMNKFTI DENDHEWKILKKLKQTTDEFRSCIEDLKVLGKKDFKMILRWRKIAREILGIEVKDDAKTEIEVVPLTEEE QIEKDLQGLQEKQRLNVKRERRRKNEMKQKELQRMQMNMITPTDIGIEAASLGKESLFNLKTAEKTGILN DLAKGKKRMIFTDDELAKDNDIYIDENIMIKDKDSAADADDLESELNAMYSDYKTRRSERDAKFRAKQAR 10 GGDNEEEWTGFNEGSLEKKEEEGKDYIEDNDDEGVEGDSDDDEAITNLISKLKGQEGDHKLSSKARMIFN DPIFNNVEPDLPVNTVNDGIMSSESVGDISKLNKKRKHEEMHQKQDEADSSDESSSDDSDFEIVANDNAS EEFDSDYDSEEEKNQTKKEKHSRDIDIATVEAMTLAHQLALGQKNKHDLVDEGFNRYTFRDTENLPDWFL EDEKEHSKINKPITKEAAMAIKEKIKAMNARPIKKVAEAKARKRMRAVARLEKIKKKAGLINDDSDKTEK DKAEEISRLMRKVTKKPKTKPKVTLVVASGRNKGLAGRPKGVKGKYKMVDGVMKNEQRALRRIAKKHHKK 15 K By “27S pre-rRNA (guanosine2922-2'-O)-methyltransferase polynucleotide” or “SPB1 polynucleotide” is meant a nucleic acid molecule encoding an SPB1 polypeptide or fragment thereof. The sequence of an exemplary SPB1 polynucleotide is provided below: >NM_001178698.1 Saccharomyces cerevisiae S288C 27S pre-rRNA (guanosine2922- 20 2'-O)-methyltransferase (SPB1), partial mRNA ATGGGTAAGACACAAAAGAAGAATAGTAAGGGACGTTTAGATAGATACTATTATCTAGCAAAGGAGAAAG GTTATCGTGCTCGTTCATCCTTTAAGATTATTCAGATTAATGAAAAGTATGGCCACTTCTTAGAAAAATC GAAGGTTGTTATTGATCTGTGTGCTGCTCCTGGTTCATGGTGTCAAGTTGCATCCAAACTCTGTCCTGTC AACTCCTTAATTATTGGTGTTGATATTGTTCCAATGAAGCCGATGCCCAATGTTATAACTTTTCAAAGTG 25 ATATTACAACAGAAGATTGTAGATCAAAATTGAGGGGTTATATGAAGACTTGGAAAGCTGATACAGTGTT GCATGATGGTGCTCCTAATGTTGGTTTGGGTTGGGTTCAGGATGCTTTCACCCAATCTCAATTAACCTTA CAAGCTTTGAAGTTGGCCGTCGAAAATTTGGTGGTGAACGGTACTTTTGTTACTAAAATTTTCAGATCCA AGGATTATAATAAATTGATTTGGGTTTTCCAACAATTGTTTGAGAAAGTTGAAGCCACAAAACCACCCGC ATCAAGAAATGTTTCTGCAGAAATTTTTGTAGTGTGTAAAGGTTTCAAGGCACCAAAGAGGCTGGACCCA 30 AGATTACTGGATCCAAAAGAAGTTTTTGAAGAATTGCCAGATGGGCAACAGAATATGGAGTCCAAGATTT ATAATCCCGAAAAAAAAGTTAGAAAAAGACAAGGTTATGAGGAAGGTGATAATTTACTGTATCATGAAAC CTCAATTTTGGATTTTGTCAGGACGGAAGACCCAATAAGCATGCTTGGAGAAATGAATAAGTTTACAATT GATGAGAATGACCATGAATGGAAGATCTTGAAGAAATTGAAACAAACCACAGACGAGTTCCGTTCCTGTA TTGAAGATTTAAAAGTCTTGGGTAAAAAGGATTTTAAAATGATCCTAAGATGGAGAAAGATTGCAAGGGA 35 AATCTTGGGCATCGAAGTTAAGGACGACGCTAAAACAGAAATTGAAGTAGTACCGTTAACAGAAGAGGAG CAAATTGAAAAAGATTTGCAAGGTTTACAGGAGAAGCAGCGTCTAAATGTCAAGCGCGAAAGAAGAAGGA AGAACGAAATGAAGCAAAAGGAACTACAGAGAATGCAAATGAACATGATAACCCCCACTGATATTGGTAT TGAAGCCGCAAGTTTGGGTAAAGAATCGTTGTTTAATTTGAAAACTGCAGAAAAGACTGGTATCTTGAAC GACTTGGCGAAGGGTAAGAAAAGAATGATTTTTACAGACGATGAATTAGCCAAAGATAATGATATTTACA 40 TTGATGAAAACATCATGATCAAAGATAAGGATTCTGCCGCTGATGCGGACGATTTAGAAAGCGAGTTGAA TGCCATGTATAGTGATTATAAAACTAGAAGGTCGGAAAGGGATGCCAAGTTTAGAGCTAAGCAAGCACGT 40 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GGTGGCGATAATGAGGAAGAATGGACCGGTTTTAATGAGGGAAGTCTAGAAAAGAAAGAAGAGGAAGGTA AAGATTATATCGAAGACAATGACGATGAAGGTGTCGAAGGAGACTCCGATGATGATGAAGCCATCACCAA TTTGATTAGCAAATTGAAGGGACAAGAAGGTGATCACAAATTGAGTAGTAAGGCACGTATGATTTTCAAT GATCCGATATTTAATAACGTTGAACCTGATTTGCCAGTAAATACCGTCAATGATGGCATAATGAGTTCCG 5 AGTCTGTTGGTGATATTTCTAAATTAAATAAGAAAAGAAAACATGAAGAAATGCACCAGAAACAAGATGA AGCAGATTCTTCGGATGAGAGTTCAAGTGATGACTCCGATTTCGAAATTGTGGCCAACGATAATGCATCA GAAGAATTCGATTCTGATTATGATTCAGAGGAAGAAAAAAATCAAACAAAGAAAGAAAAGCATTCCAGAG ACATTGATATTGCTACTGTTGAAGCCATGACTTTGGCACATCAGTTAGCATTGGGTCAGAAAAACAAGCA TGATCTTGTTGATGAAGGTTTCAATAGATACACCTTCCGTGACACCGAAAATTTGCCAGATTGGTTTTTA 10 GAAGATGAAAAGGAACATTCAAAGATAAATAAGCCGATTACTAAGGAGGCAGCGATGGCAATTAAAGAAA AAATAAAGGCAATGAACGCCCGTCCTATTAAGAAGGTTGCTGAAGCTAAGGCAAGAAAGAGGATGCGTGC CGTAGCTCGGTTAGAAAAGATCAAGAAGAAGGCGGGCTTGATTAACGATGATTCAGACAAGACAGAGAAG GACAAGGCTGAAGAAATTTCTAGATTGATGCGTAAGGTTACCAAAAAACCAAAGACCAAGCCAAAGGTTA CTTTGGTTGTTGCCTCAGGTAGGAACAAAGGTTTGGCAGGTAGACCAAAGGGCGTTAAGGGTAAGTATAA 15 GATGGTTGATGGTGTCATGAAGAATGAACAAAGAGCCCTAAGACGTATCGCAAAGAAGCATCACAAGAAA AAGTAG By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Ranges provided herein are understood to be shorthand for all of the values within the 20 range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. By “tRNA guanosine 2 -O-methyltransferase polypeptide” or “TARBP1 polypeptide” is 25 meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession No. NP_005637.3, or a fragment thereof, and having methyltransferase activity. The sequences of an exemplary TARBP1 polypeptide is provided below: >NP_005637.3 probable methyltransferase TARBP1 [Homo sapiens] MEWVLAEALLSQSRDPRALLGALCQGEASAERVETLRFLLQRLEDEEARGSGGAGALPEAAREVAAGYLV 30 PLLRSLRGRPAGGPDPSLQPRHRRRVLRAAGAALRSCVRLAGRPQLAAALAEEALRDLLAGWRAPGAEAA VEVLAAVGPCLRPREDGPLLERVAGTAVALALGGGGDGDEAGPAEDAAALVAGRLLPVLVQCGGAALRAV WGGLAAPGASLGSGRVEEKLLVLSALAEKLLPEPGGDRARGAREAGPDARRCWRFWRTVQAGLGQADALT RKRARYLLQRAVEVSAELGADCTCGPQEGNGPSLFWWSERKKDELLKFWENYILIMETLEGNQIHVIKPV LPKLNNLFEYAVSEENGCWLFHPSWHMCIYKRMFESENKILSKEGVIHFLELYETKILPFSPEFSEFIIG 35 PLMDALSESSLYSRSPGQPIGSCSPLGLKLQKFLVTYISLLPEEIKSSFLLKFIRKMTSRHWCAVPILFL SKALANVPRHKALGIDGLLALRDVIHCTMITHQILLRGAAQCYLLQTAMNLLDVEKVSLSDVSTFLMSLR QEESLGRGTSLWTELCDWLRVNESYFKPSPTCSSIGLHKTSLNAYVKSIVQEYVKSSAWETGENCFMPDW FEAKLVSLMVLLAVDVEGMKTQYSGKQRTENVLRIFLDPLLDVLMKFSTNAYMPLLKTDRCLQLLLKLLN TCRLKGSSAQDDEVSTVLQNFFMSTTESISEFILRRLTMNELNSVSDLDRCHLYLMVLTELINLHLKVGW 41 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 KRGNPIWRVISLLKNASIQHLQEMDSGQEPTVGSQIQRVVSMAALAMVCEAIDQKPELQLDSLHAGPLES FLSSLQLNQTLQKPHAEEQSSYAHPLECSSVLEESSSSQGWGKIVAQYIHDQWVCLSFLLKKYHTLIPTT GSEILEPFLPAVQMPIRTLQSALEALTVLSSDQVLPVFHCLKVLVPKLLTSSESLCIESFDMAWKIISSL SNTQLIFWANLKAFVQFVFDNKVLTIAAKIKGQAYFKIKEIMYKIIEMSAIKTGVFNTLISYCCQSWIVS 5 ASNVSQGSLSSAKNYSELILEACIFGTVFRRDQRLVQDVQTFIENLGHDCAANIVMENTKREDHYVRICA VKFLCLLDGSNMSHKLFIEDLAIKLLDKDELVSKSKKRYYVNSLQHRVKNRVWQTLLVLFPRLDQNFLNG IIDRIFQAGFTNNQASIKYFIEWIIILILHKFPQFLPKFWDCFSYGEENLKTSICTFLAVLSHLDIITQN IPEKKLILKQALIVVLQWCFNHNFSVRLYALVALKKLWTVCKVLSVEEFDALTPVIESSLHQVESMHGAG NAKKNWQRIQEHFFFATFHPLKDYCLETIFYILPRLSGLIEDEWITIDKFTRFTDVPLAAGFQWYLSQTQ 10 LSKLKPGDWSQQDIGTNLVEADNQAEWTDVQKKIIPWNSRVSDLDLELLFQDRAARLGKSISRLIVVASL IDKPTNLGGLCRTCEVFGASVLVVGSLQCISDKQFQHLSVSAEQWLPLVEVKPPQLIDYLQQKKTEGYTI IGVEQTAKSLDLTQYCFPEKSLLLLGNEREGIPANLIQQLDVCVEIPQQGIIRSLNVHVSGALLIWEYTR QQLLSHGDTKP 15 By “tRNA guanosine 2 -O-methyltransferase polynucleotide” or “TARBP1 polynucleotide” is meant a nucleic acid molecule encoding an TARBP1 polypeptide or fragment thereof. The sequence of an exemplary TARBP1 polynucleotide is provided below: >NM_005646.4 Homo sapiens tRNA guanosine 2 -O-methyltransferase TARBP1 (TARBP1), mRNA 20 CGGTCCTGTGCGCACGCATCGCACACGCCGGCGCCTTCCTTTGGGAGCCCGGGCCGGTGGCGCGGGCGCT CGGCAAATGGAGTGGGTGCTCGCGGAAGCGCTGCTCTCGCAGAGCCGGGACCCCCGGGCCCTGCTTGGGG CGCTGTGCCAAGGGGAGGCATCCGCGGAGCGCGTGGAGACGCTGCGCTTCCTTCTGCAGCGGCTCGAGGA CGAGGAGGCGCGCGGCAGCGGGGGCGCAGGCGCGCTCCCGGAGGCGGCGCGCGAGGTGGCTGCAGGGTAC CTCGTGCCACTGCTGCGGAGCCTGCGCGGACGCCCCGCGGGCGGCCCGGACCCCAGTCTGCAGCCTCGCC 25 ACCGCCGGCGCGTGCTGAGGGCGGCGGGCGCGGCCCTGCGCTCGTGCGTCCGCCTGGCCGGGCGTCCGCA GCTGGCGGCCGCGCTGGCTGAGGAGGCGCTGCGCGATCTGCTCGCCGGGTGGCGCGCGCCTGGCGCCGAG GCTGCCGTGGAAGTGCTAGCAGCCGTCGGGCCATGTTTGCGGCCCCGCGAGGACGGGCCGCTACTGGAGC GCGTGGCGGGGACCGCCGTCGCCCTGGCGCTGGGCGGGGGCGGGGACGGGGATGAGGCCGGGCCTGCCGA GGACGCGGCGGCGCTGGTGGCCGGGCGACTGCTGCCAGTGCTGGTCCAATGTGGCGGGGCGGCGCTGCGG 30 GCCGTGTGGGGCGGGCTGGCCGCGCCTGGGGCGTCCCTGGGGTCCGGCCGCGTAGAGGAGAAGCTGCTGG TCCTGAGCGCCCTGGCCGAGAAGCTGTTGCCCGAGCCCGGCGGCGACCGCGCCCGCGGCGCGCGCGAGGC GGGCCCGGACGCCCGGCGCTGCTGGCGCTTCTGGAGGACGGTGCAGGCGGGGCTGGGCCAGGCGGACGCC CTGACGCGCAAGCGAGCGCGCTACCTGCTGCAGAGGGCGGTGGAGGTGTCGGCGGAGCTGGGGGCCGACT GCACCTGCGGGCCCCAGGAAGGAAACGGCCCAAGTCTGTTTTGGTGGTCTGAGAGGAAAAAAGATGAGCT 35 TCTAAAGTTTTGGGAAAATTATATTTTAATTATGGAGACTTTAGAAGGAAATCAGATACATGTTATAAAG CCAGTTTTACCAAAGCTAAACAATCTGTTTGAATATGCGGTGTCAGAGGAAAATGGATGTTGGCTCTTTC ACCCATCCTGGCATATGTGTATTTATAAAAGAATGTTTGAAAGTGAAAACAAAATCCTGTCCAAAGAAGG TGTTATCCATTTTTTGGAGCTGTATGAAACAAAGATTCTTCCATTTTCACCAGAATTTTCTGAGTTTATT ATTGGACCATTAATGGATGCGCTTTCAGAGAGCTCTCTGTATAGCAGGTCCCCAGGCCAGCCAATAGGAA 40 GCTGTTCTCCATTGGGACTGAAATTACAGAAGTTTTTAGTCACTTATATTTCTCTTCTTCCAGAAGAAAT 42 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 AAAGAGTAGCTTCCTATTGAAGTTTATTCGGAAGATGACAAGTAGGCATTGGTGTGCTGTTCCCATTTTG TTTCTATCTAAGGCTTTGGCAAATGTCCCAAGACATAAGGCCCTGGGTATAGATGGGCTTCTTGCTCTCA GGGATGTTATTCATTGCACTATGATCACACATCAGATTCTCCTGAGAGGGGCAGCCCAATGCTACCTTCT TCAAACAGCTATGAATTTGCTAGATGTGGAGAAAGTGTCACTTTCTGATGTCTCAACTTTTCTCATGTCT 5 CTGAGACAAGAGGAATCCTTAGGACGAGGAACTTCATTGTGGACAGAGCTGTGTGACTGGCTACGTGTTA ATGAAAGCTATTTTAAGCCATCCCCTACGTGTAGCTCCATTGGACTTCACAAGACATCTTTAAATGCTTA TGTAAAGAGCATTGTTCAAGAGTATGTTAAGTCATCTGCTTGGGAAACAGGAGAAAACTGCTTTATGCCT GATTGGTTTGAAGCCAAGCTTGTTTCTCTGATGGTCTTGCTGGCTGTGGATGTGGAAGGAATGAAGACTC AGTATAGCGGAAAGCAGAGAACAGAGAATGTATTGCGGATATTCTTAGACCCTCTTCTGGATGTGCTTAT 10 GAAGTTTAGTACCAATGCCTACATGCCCTTGCTGAAGACTGACAGATGCCTCCAGCTGCTGTTGAAGCTG TTGAACACATGCAGGTTGAAAGGTTCCAGTGCCCAAGATGATGAGGTGTCTACTGTTCTTCAGAACTTTT TCATGTCTACTACAGAGAGCATTTCTGAATTTATTCTCAGAAGACTTACTATGAATGAGCTAAATAGTGT TTCAGATCTGGATCGTTGCCATTTATACCTGATGGTGTTAACTGAGCTTATAAATCTGCATTTGAAGGTT GGGTGGAAAAGGGGTAACCCTATCTGGAGAGTTATTTCTCTTTTGAAAAATGCATCCATTCAGCATCTTC 15 AAGAGATGGACAGTGGACAGGAGCCAACAGTTGGAAGTCAGATTCAGAGAGTAGTGAGCATGGCTGCCTT GGCCATGGTGTGTGAGGCCATAGACCAGAAGCCTGAGCTGCAGCTGGACTCTCTCCATGCTGGGCCCCTG GAAAGCTTCCTTTCCTCTCTTCAGCTCAATCAGACGCTGCAGAAGCCCCACGCAGAGGAGCAGAGCAGTT ATGCTCACCCCTTGGAGTGCAGCAGTGTTTTGGAAGAATCGTCATCTTCCCAAGGATGGGGAAAAATAGT TGCACAATATATTCATGATCAATGGGTGTGCCTCTCTTTCCTGTTGAAAAAATATCACACCCTTATACCA 20 ACCACAGGGAGTGAAATTCTGGAACCGTTTCTACCTGCCGTTCAGATGCCAATAAGGACTTTGCAGTCTG CACTAGAAGCCCTCACAGTTCTTTCTTCTGATCAAGTTTTACCAGTGTTCCATTGCTTGAAAGTGTTGGT TCCCAAGCTTCTGACTTCCTCTGAATCACTCTGCATAGAGTCTTTTGACATGGCGTGGAAAATTATATCT TCTTTAAGCAACACTCAGCTGATATTCTGGGCTAATTTAAAAGCTTTTGTTCAGTTTGTTTTTGATAACA AAGTTCTTACCATTGCTGCCAAAATCAAGGGCCAGGCATATTTCAAAATAAAAGAGATTATGTACAAGAT 25 AATTGAAATGTCTGCTATAAAGACTGGAGTCTTCAATACACTGATAAGTTACTGCTGTCAGTCTTGGATA GTGTCTGCTTCAAATGTGTCCCAAGGATCTTTATCAAGTGCTAAAAATTATAGCGAACTTATCCTTGAGG CTTGTATATTTGGAACTGTGTTTAGGCGTGATCAAAGACTTGTTCAGGATGTACAGACCTTCATAGAAAA CCTTGGACATGACTGTGCGGCAAATATTGTTATGGAAAATACTAAGAGAGAAGACCATTATGTGAGAATT TGTGCTGTCAAATTCCTGTGTTTATTAGATGGCTCCAATATGTCCCACAAGTTGTTTATTGAGGATCTTG 30 CAATCAAGCTATTAGATAAAGATGAATTAGTGTCCAAGTCCAAAAAACGCTACTATGTGAATTCTCTACA GCACAGAGTGAAAAACCGAGTGTGGCAGACTCTGCTGGTACTTTTCCCTAGACTTGACCAGAATTTCTTG AATGGAATTATTGACAGGATTTTCCAGGCTGGTTTCACCAACAATCAAGCATCCATAAAATATTTTATAG AATGGATTATTATATTGATTCTTCATAAATTCCCTCAATTTCTTCCAAAGTTCTGGGATTGTTTTTCTTA TGGTGAAGAAAATCTTAAAACAAGCATTTGTACGTTTTTAGCAGTTTTATCACATTTAGACATTATTACT 35 CAAAATATTCCAGAAAAGAAACTAATTCTGAAGCAAGCCCTTATAGTTGTGCTGCAGTGGTGTTTCAATC ACAATTTTAGTGTTCGACTGTATGCTTTAGTTGCTCTTAAGAAACTCTGGACTGTGTGTAAAGTGTTAAG TGTTGAAGAATTTGATGCCCTGACTCCTGTGATTGAATCCAGCCTCCATCAAGTGGAAAGCATGCACGGA GCAGGGAATGCCAAGAAGAATTGGCAACGCATTCAGGAGCATTTCTTTTTTGCAACATTTCACCCACTCA AGGATTATTGTCTAGAGACCATATTTTACATCCTTCCACGCCTTTCAGGCCTTATTGAAGATGAATGGAT 40 CACCATTGATAAATTTACCAGATTCACTGATGTTCCTTTAGCTGCGGGATTTCAGTGGTACCTTTCTCAA ACTCAACTTAGTAAACTAAAACCAGGTGACTGGTCTCAGCAAGACATAGGTACTAATTTGGTCGAAGCAG 43 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ATAACCAAGCGGAGTGGACCGACGTTCAGAAGAAGATTATCCCGTGGAACAGTCGTGTTTCCGACTTAGA CCTGGAGCTCCTGTTTCAGGATCGTGCTGCCAGACTTGGAAAGTCAATTAGTAGACTCATCGTTGTGGCC TCGCTCATCGACAAACCGACCAATTTAGGAGGACTGTGCAGGACCTGTGAGGTATTTGGGGCTTCAGTGC TCGTTGTTGGCAGCCTTCAGTGTATCAGCGACAAACAGTTTCAGCACCTCAGTGTCTCTGCAGAACAGTG 5 GCTTCCTCTAGTGGAGGTAAAACCACCTCAGCTAATTGATTATCTGCAGCAGAAGAAAACAGAAGGTTAT ACCATCATTGGAGTGGAACAAACTGCCAAAAGTTTAGACCTAACCCAATATTGCTTTCCTGAGAAATCTC TGCTCTTGTTGGGAAATGAACGTGAGGGAATTCCAGCAAATCTGATCCAACAGTTGGACGTTTGTGTGGA AATTCCTCAACAGGGCATTATCCGCTCCCTGAATGTCCATGTGAGTGGAGCCCTGCTGATCTGGGAGTAC ACCAGGCAGCAGCTGCTCTCGCACGGAGATACCAAGCCATGATGTGCCTTCCTTAGTGAACTGCTGCTGC 10 TGTTCAGACTTTTTTAAAAAAAACTATTTGGACTAAAGAAACAGATTCTGAAATTTATTGTGATAATTTG TATTTCTTTTTTCTTGCAATTTAATGCCAAAAGTTTGCCATGTGCCTTAAACATATTACTATATATTTTC CCCTTTAATAAACACTTTTTGTTAAATTGTATTCTTCCTTTAATAAAATATTTTAAGCAATTGTGGAAAT AAAACAATGGATTTTTTAAGAGAGAA 15 As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. Unless specifically stated or obvious from context, as used herein, the term "or" is 20 understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural. By “tRNA methyltransferase 11 homolog polypeptide” or “TRMT11 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001337526.1, NP_001337525.1, NP_001337524.1, NP_001337523.1, 25 NP_001337522.1, NP_001337521.1, NP_001337520.1, NP_001337519.1, NP_001337518.1, NP_001337517.1, NP_001337516.1, NP_001337515.1, NP_001337514.1, NP_001337513.1, NP_001337512.1, NP_001337511.1, NP_001337510.1, NP_001337509.1, or NP_001026882.2, or a fragment thereof, and having methyltransferase activity. The sequences of exemplary TRMT11 polypeptides are provided below: 30 >NP_001026882.2 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=a] MALSCTLNRYLLLMAQEHLEFRLPEIKSLLLLFGGQFASSQETYGKSPFWILSIPSEDIARNLMKRTVCA KSIFELWGHGQSPEELYSSLKNYPVEKMVPFLHSDSTYKIKIHTFNKTLTQEEKIKRIDALEFLPFEGKV NLKKPQHVFSVLEDYGLDPNCIPENPHNIYFGRWIADGQRELIESYSVKKRHFIGNTSMDAGLSFIMANH GKVKENDIVFDPFVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDENIRANLRQYGLE 35 KYYLDVLVSDASKPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLS DMFLDLLNFAAETLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFE NRDQYSHLLSDHFLPYQGHNSFREKYFSGVTKRIAKEEKSTQE 44 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_001337509.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=b] MALSCTLNRYLLLMAQEHLEFRLPFASSQETYGKSPFWILSIPSEDIARNLMKRTVCAKSIFELWGHGQS PEELYSSLKNYPVEKMVPFLHSDSTYKIKIHTFNKTLTQEEKIKRIDALEFLPFEGKVNLKKPQHVFSVL EDYGLDPNCIPENPHNIYFGRWIADGQRELIESYSVKKRHFIGNTSMDAGLSFIMANHGKVKENDIVFDP 5 FVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDAS KPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAE TLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDH FLPYQGHNSFREKYFSGVTKRIAKEEKSTQE 10 >NP_001337510.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=c] MKRTVCAKSIFELWGHGQSPEELYSSLKNYPVEKMVPFLHSDSTYKIKIHTFNKTLTQEEKIKRIDALEF LPFEGKVNLKKPQHVFSVLEDYGLDPNCIPENPHNIYFGRWIADGQRELIESYSVKKRHFIGNTSMDAGL SFIMANHGKVKENDIVFDPFVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDENIRAN LRQYGLEKYYLDVLVSDASKPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPV 15 SLSYHLSDMFLDLLNFAAETLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITM EKVKKFENRDQYSHLLSDHFLPYQGHNSFREKYFSGVTKRIAKEEKSTQE >NP_001337511.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=c] MKRTVCAKSIFELWGHGQSPEELYSSLKNYPVEKMVPFLHSDSTYKIKIHTFNKTLTQEEKIKRIDALEF 20 LPFEGKVNLKKPQHVFSVLEDYGLDPNCIPENPHNIYFGRWIADGQRELIESYSVKKRHFIGNTSMDAGL SFIMANHGKVKENDIVFDPFVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDENIRAN LRQYGLEKYYLDVLVSDASKPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPV SLSYHLSDMFLDLLNFAAETLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITM EKVKKFENRDQYSHLLSDHFLPYQGHNSFREKYFSGVTKRIAKEEKSTQE 25 >NP_001337512.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=c] MKRTVCAKSIFELWGHGQSPEELYSSLKNYPVEKMVPFLHSDSTYKIKIHTFNKTLTQEEKIKRIDALEF LPFEGKVNLKKPQHVFSVLEDYGLDPNCIPENPHNIYFGRWIADGQRELIESYSVKKRHFIGNTSMDAGL SFIMANHGKVKENDIVFDPFVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDENIRAN 30 LRQYGLEKYYLDVLVSDASKPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPV SLSYHLSDMFLDLLNFAAETLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITM EKVKKFENRDQYSHLLSDHFLPYQGHNSFREKYFSGVTKRIAKEEKSTQE >NP_001337513.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=c] 35 MKRTVCAKSIFELWGHGQSPEELYSSLKNYPVEKMVPFLHSDSTYKIKIHTFNKTLTQEEKIKRIDALEF LPFEGKVNLKKPQHVFSVLEDYGLDPNCIPENPHNIYFGRWIADGQRELIESYSVKKRHFIGNTSMDAGL SFIMANHGKVKENDIVFDPFVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDENIRAN LRQYGLEKYYLDVLVSDASKPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPV SLSYHLSDMFLDLLNFAAETLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITM 40 EKVKKFENRDQYSHLLSDHFLPYQGHNSFREKYFSGVTKRIAKEEKSTQE 45 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_001337514.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=d] MDAGLSFIMANHGKVKENDIVFDPFVGTGGLLIACAHFGAYVYGTDIDYNTVHGLGKATRKNQKWRGPDE NIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFDAIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPE SHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYWLPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSR 5 RLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFREKYFSGVTKRIAKEEKSTQE >NP_001337515.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW 10 LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE >NP_001337516.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD 15 AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE >NP_001337517.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] 20 MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE 25 >NP_001337518.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE 30 >NP_001337519.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR 35 EKYFSGVTKRIAKEEKSTQE >NP_001337520.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW 40 LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE 46 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NP_001337521.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW 5 LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE >NP_001337522.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD 10 AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE >NP_001337523.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] 15 MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE 20 >NP_001337524.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE 25 >NP_001337525.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR 30 EKYFSGVTKRIAKEEKSTQE >NP_001337526.1 TRMT11 [organism=Homo sapiens] [GeneID=60487] [isoform=e] MTENKNTLFNTFSLPFSFPSGKATRKNQKWRGPDENIRANLRQYGLEKYYLDVLVSDASKPSWRKGTYFD AIITDPPYGIRESTRRTGSQKEIPKGIEKWEKCPESHVPVSLSYHLSDMFLDLLNFAAETLVLGGRLVYW 35 LPVYTPEYTEEMVPWHPCLELVSNCEQKLSSHTSRRLITMEKVKKFENRDQYSHLLSDHFLPYQGHNSFR EKYFSGVTKRIAKEEKSTQE By “tRNA methyltransferase 11 homolog polynucleotide” or “TRMT11 polynucleotide” is meant a nucleic acid molecule encoding an TRMT11 polypeptide or fragment thereof. The 40 sequences of exemplary TRMT11 polynucleotides are provided below: 47 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_001031712.3 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 1, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT 5 TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCT TAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCAC ACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTG AAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAA 10 CTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATT GAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTA TGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCT GATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTG GGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAAT 15 ATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCAC ATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAG AAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTT ATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACT AGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAA 20 CTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGA AGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAA TTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAA TGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTT TCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAG 25 CAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTAT TTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATC TACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTT AAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATA AACCATATTGAGAATTTCTAAAA 30 >NM_001350580.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 2, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGTTTGCCAGCAGTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCC 35 TCTGAAGATATTGCAAGAAATTTGATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTC ATGGACAATCTCCTGAGGAGCTGTACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCT ACATTCGGACTCTACATATAAAATAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATC AAGCGAATAGATGCACTTGAATTTCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTAT TTTCTGTTTTGGAGGATTATGGTTTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGG 40 TAGATGGATTGCAGATGGACAGAGAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGA AATACAAGTATGGATGCTGGTTTGTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTG 48 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TCTTTGATCCATTTGTTGGAACAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATGTGTATGG GACAGACATAGACTACAACACAGTTCATGGCTTGGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGA CCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTT CAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGG 5 TATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAA TGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACT TCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACAC TGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCAC ACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGC 10 TAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAA AAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAG AATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAA TATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTT TTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGA 15 TAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACAT CTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGT TTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350581.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 20 transcript variant 3, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA 25 GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT GATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTG TACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAA TAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATT TCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGT 30 TTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGA GAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTT GTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACA GGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAG TTCATGGCTTGGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAA 35 TCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGG AGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAA CAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGT TTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTA GGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACC 40 CTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAAT GGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATAC 49 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAAT CCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTG GATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAA AGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTT 5 TATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTT TACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAAT TAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACAT TGCCACTAATAAACCATATTGAGAATTTCTAAAA 10 >NM_001350582.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 4, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTCAGTTTGCCAGCAGTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCA TTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATG 15 GGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCA TTTCTACATTCGGACTCTACATATAAAATAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGA AAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACA TGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTAT TTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTA 20 TTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGA TATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATGTG TATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGGAAAGGCTACTAGGAAAAACCAGAAGTGGA GAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCT GGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCA 25 TATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGG AAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTT AAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAA TACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCA GTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACA 30 TCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTA ACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGA ATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTT TAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAG ACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGAT 35 GGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAA AACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTAT TTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350583.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 40 transcript variant 5, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA 50 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGTTTGCCAGCAGTCAAGAAACTTATGGAAAGTCACCATTTTGGATTC TTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGACAGTGTGTGCCAAGTCTATATTTGA 5 ACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATG GTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCACACTTTTAATAAGACATTGACACAAG AAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACC GCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAACTGCATCCCTGAGAATCCACATAAT ATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGAC 10 ACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTATGGCTAACCATGGAAAAGTGAAAGA AAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCA TATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGGAAAGGCTACTAGGAAAAACCAGA AGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGA TGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGAT 15 CCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAA AATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGA CCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACG CCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGC TTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTA 20 TTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGT GGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAA GAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTA CGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTT TTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGT 25 ACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGAC TTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATAT GTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350584.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 30 transcript variant 6, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACTTA TGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGACA 35 GTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCTTA AAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCACAC TTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTGAA GGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAACT GCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATTGA 40 GTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTATG GCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCTGA 51 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGG AAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATAT GGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACAT ATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAA 5 GGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTAT CATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAG TCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACT CGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAG AAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATT 10 CCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATG AAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTC ATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCA ATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTT AATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTA 15 CAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAA GAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAA CCATATTGAGAATTTCTAAAA >NM_001350585.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 20 transcript variant 7, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA 25 GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT GATGAAACGGACAGTGTGTGCCAAGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCACA CTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTGA AGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAAC TGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATTG 30 AGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTAT GGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCTG ATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGG GAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATA TGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACA 35 TATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGA AGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTA TCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTA GTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAAC TCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAA 40 GAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAAT TCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAAT 52 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTT CATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGC AATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATT TAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCT 5 ACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTA AGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAA ACCATATTGAGAATTTCTAAAA >NM_001350586.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 10 transcript variant 8, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCT 15 TAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCAC ACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTG AAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAA CTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATT GAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTA 20 TGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCT GATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTG GGACTGTTTACTGCTGGAAGAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTAC TTGAAAATATCGAATGAGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACAT CAGTACATTTTACTGTTATGTTGAAATTAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAA 25 TGAAAGCAAATTTTGAATAAATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAAT AAGAATACTCTGTTTAATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACC AGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCT TGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACT GATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAG 30 AAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCT TGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTAT ACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGA AGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCA GTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTT 35 AGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAAT GAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAG GTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTT GTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCA AGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTT 40 GACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGA TATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAA 53 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 A >NM_001350587.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 9, mRNA 5 GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT 10 GATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTG TACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAA TAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATT TCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGT TTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGA 15 GAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTT GTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACA GTGTACTTGTAAATTGGGTCTTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAGCAC AGGAAAGCTTTTCTGTTCCCCTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCATTTT GGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGGACTGTTTACTGCTGGAA 20 GAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAATGAGT GTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTGTTAT GTTGAAATTAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTGAATA AATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTTAATA CTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGA 25 TGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGAT GCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCA GAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCC AGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCA GCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAG 30 AGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATC AAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGT GATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAA TTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTT GATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTT 35 TATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGT TGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGAC TCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCT GGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTG GTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA 40 54 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_001350588.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 10, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG 5 AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT GATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTG TACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAA 10 TAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATT TCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGT TTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGA GAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTT GTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACA 15 GGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAG TTCATGGCTTGGGACTGTTTACTGCTGGAAGAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAA AATCTTCGTACTTGAAAATATCGAATGAGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGAT AAGTTAGACATCAGTACATTTTACTGTTATGTTGAAATTAAAATAACTATTTACTTGTATACATAAAATT AAGATTTTAAATGAAAGCAAATTTTGAATAAATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAA 20 TGACAGAAAATAAGAATACTCTGTTTAATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTAC TAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAG AAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATG CAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACC AAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGT 25 GATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGT TACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAA CTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAG AATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTG AGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAG 30 ATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGAT CCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTA AAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATAT TTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAA TTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATT 35 CATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGA GAATTTCTAAAA >NM_001350589.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 11, mRNA 40 GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT 55 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCT TAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCAC ACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTG 5 AAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAA CTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATT GAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTA TGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGCAGTGTACTT GTAAATTGGGTCTTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAGCACAGGAAAGC 10 TTTTCTGTTCCCCTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATA TGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGCCTGCTCAACATGAAGGTGATGAGGA TGAAGACCTTTATGATGATCCACTTCCACTTCATGAATAGTAAATCTATTTTTACTGAATAAAGACTGTT TACTGCTGGAAGAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAAT ATCGAATGAGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACAT 15 TTTACTGTTATGTTGAAATTAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCA AATTTTGAATAAATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAATAAGAATAC TCTGTTTAATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGG AGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCC TGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCC 20 ATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGG GAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGT TAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGA ATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCC AGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCAC 25 ATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGT AACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAG AATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTT TTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATA GACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGA 30 TGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTA AAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTA TTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350590.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 35 transcript variant 12, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCT 40 TAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCAC ACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTG 56 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 AAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAA CTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATT GAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTA TGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCT 5 GATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTG GCCTGCTCAACATGAAGGTGATGAGGATGAAGACCTTTATGATGATCCACTTCCACTTCATGAATAGTAA ATCTATTTTTACTGAATAAAGCGTCTACCTATCTTTGAGCACTTCTCCGTCACACTGCATGTTTTTCAGA GTCATGTCGTTCTTCTTTTGATTTTATAAGAATAATTATAGAGCAAACATGTAACTGCCTCCTAAGTGAA CAAAAAGAATGTTATTCAGCCTTATGGAGTCAGTATAAGATTATATGCTGATAACTCCAAAGTCTGTATA 10 TTCTGGCCTTCAACTACCTGCTGAATGTCCCCACTGCGTATCTTTTTCTGAAAGTTCTGCAGGTACCTCA AGGACTGTTTACTGCTGGAAGAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTA CTTGAAAATATCGAATGAGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACA TCAGTACATTTTACTGTTATGTTGAAATTAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAA ATGAAAGCAAATTTTGAATAAATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAA 15 TAAGAATACTCTGTTTAATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAAC CAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACC TTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTAC TGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATA GAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTC 20 TTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTA TACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAG AAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACC AGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTT TAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAA 25 TGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATA GGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTT TGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTC AAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGT TGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAG 30 ATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAA AA >NM_001350591.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 13, mRNA 35 GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCT TAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCAC 40 ACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTG AAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAA 57 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATT GAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTA TGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGGTGGCCTGCT GATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTG 5 GCCTGCTCAACATGAAGGTGATGAGGATGAAGACCTTTATGATGATCCACTTCCACTTCATGAATAGTAA ATCTATTTTTACTGAATAAAGACTGTTTACTGCTGGAAGAAAGGAACCATATTCTCTTCAACTCTATCTT ACTACAAAAATCTTCGTACTTGAAAATATCGAATGAGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAG AACAAGATAAGTTAGACATCAGTACATTTTACTGTTATGTTGAAATTAAAATAACTATTTACTTGTATAC ATAAAATTAAGATTTTAAATGAAAGCAAATTTTGAATAAATGTAGGTTTCAGTGAGTTAAAACATTACTT 10 TTCCTAAATGACAGAAAATAAGAATACTCTGTTTAATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGA AAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCTTCGTCAATATG GTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGGAAGGGCACATA TTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAG GAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATC 15 ATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGT CTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTC GTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGA AATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAAGGTCATAATTC CTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGA 20 AAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGATGTGAACTTTCA TGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAA TGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTATAATCTTATTTA ATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTACAATTAATCTAC AAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAATAGTAGTTAAG 25 AATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGCCACTAATAAAC CATATTGAGAATTTCTAAAA >NM_001350592.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 14, mRNA 30 GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA CAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCT TAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCAC 35 ACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTG AAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAA CTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATT GAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTA TGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGCAGTGTACTT 40 GTAAATTGGGTCTTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAGCACAGGAAAGC TTTTCTGTTCCCCTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATA 58 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGGACTGTTTACTGCTGGAAGAAAGGAA CCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAATGAGTGTTTTAAG GCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTGTTATGTTGAAAT TAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTGAATAAATGTAGG 5 TTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTTAATACTTTCTCT CTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACA TTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAA ACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCT ACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCC 10 ATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGAC CCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTG CCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCT TGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTT TCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAG 15 GAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAA AAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGA AAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTA TTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACA GTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATAT 20 TATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCAT CGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350593.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 15, mRNA 25 GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT 30 GATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTG TACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAA TAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATT TCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGT TTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGA 35 GAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTT GTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACA GCAGTGTACTTGTAAATTGGGTCTTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAG CACAGGAAAGCTTTTCTGTTCCCCTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCAT TTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGCCTGCTCAACATGAA 40 GGTGATGAGGATGAAGACCTTTATGATGATCCACTTCCACTTCATGAATAGTAAATCTATTTTTACTGAA TAAAGCGTCTACCTATCTTTGAGCACTTCTCCGTCACACTGCATGTTTTTCAGAGTCATGTCGTTCTTCT 59 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TTTGATTTTATAAGAATAATTATAGAGCAAACATGTAACTGCCTCCTAAGTGAACAAAAAGAATGTTATT CAGCCTTATGGAGTCAGTATAAGATTATATGCTGATAACTCCAAAGTCTGTATATTCTGGCCTTCAACTA CCTGCTGAATGTCCCCACTGCGTATCTTTTTCTGAAAGTTCTGCAGGTACCTCAAGGACTGTTTACTGCT GGAAGAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAAT 5 GAGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTG TTATGTTGAAATTAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTG AATAAATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTT AATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGAC CAGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTC 10 AGATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGT ATCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAAT GTCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTT CGCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACT GAAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACA 15 CATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCT AAGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAA AGAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGA ATTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAAT ATTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTT 20 TTGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGAT AGACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATC TGCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTT TTTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA 25 >NM_001350594.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 16, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACT TATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGA 30 CAGTGTGTGCCAAGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCACACTTTTAATAAG ACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTGAAGGAAAAGTGA ATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAACTGCATCCCTGA GAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATTGAGTCATACAGT GTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTATGGCTAACCATG 35 GAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGCAGTGTACTTGTAAATTGGGTC TTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAGCACAGGAAAGCTTTTCTGTTCCC CTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGAC AGACATAGACTACAACACAGTTCATGGCTTGGGACTGTTTACTGCTGGAAGAAAGGAACCATATTCTCTT CAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAATGAGTGTTTTAAGGCAGTTTTACTT 40 GGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTGTTATGTTGAAATTAAAATAACTAT TTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTGAATAAATGTAGGTTTCAGTGAGTT 60 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 AAAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTTAATACTTTCTCTCTTCCTTTTTCT TTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATC TTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAG GAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACA 5 GGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTT CCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGG TGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCT TGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGG AAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCA 10 AGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCC ACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGA TGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAG TAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTA TAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTA 15 CAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTA ATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTG CCACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350595.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), 20 transcript variant 17, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCAGTCAAGAAACTTA TGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTTGATGAAACGGACA 25 GTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTGTACAGTTCTCTTA AAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAATAAAGATTCACAC TTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATTTCTGCCATTTGAA GGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGTTTAGACCCAAACT GCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGAGAGAGCTTATTGA 30 GTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTTGTCATTCATTATG GCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACAGCAGTGTACTTGT AAATTGGGTCTTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAGCACAGGAAAGCTT TTCTGTTCCCCTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATG TGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGGACTGTTTACTGCTGGAAGAAAGGAACC 35 ATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAATGAGTGTTTTAAGGC AGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTGTTATGTTGAAATTA AAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTGAATAAATGTAGGTT TCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTTAATACTTTCTCTCT TCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATT 40 AGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAAC CTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTAC 61 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 AAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCAT GTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCC TCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCC TTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTG 5 ATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTC TGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGA AGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAA GACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAA AGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATT 10 ACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGT TATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTA TATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCG ATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA 15 >NM_001350596.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 18, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC 20 ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT GATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTG TACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAA TAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATT 25 TCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGT TTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGA GAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTT GTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACA GGTGGCCTGCTGATAGCATGTGCTCATTTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAG 30 TTCATGGCTTGGCCTGCTCAACATGAAGGTGATGAGGATGAAGACCTTTATGATGATCCACTTCCACTTC ATGAATAGTAAATCTATTTTTACTGAATAAAGACTGTTTACTGCTGGAAGAAAGGAACCATATTCTCTTC AACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAATGAGTGTTTTAAGGCAGTTTTACTTG GAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTGTTATGTTGAAATTAAAATAACTATT TACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTGAATAAATGTAGGTTTCAGTGAGTTA 35 AAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTTAATACTTTCTCTCTTCCTTTTTCTT TCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACCAGATGAAAACATTAGGGCCAATCT TCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCAGATGCATCTAAACCTTCCTGGAGG AAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTATCAGAGAATCTACAAGAAGAACAG GTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATGTCCAGAAAGCCATGTTCCTGTTTC 40 CTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTCGCAGCTGAGACCCTCGTTTTAGGT GGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTGAAGAGATGGTGCCTTGGCACCCTT 62 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACACATCAAGGCGCTTGATCACAATGGA AAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTAAGTGATCATTTTCTGCCATACCAA GGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAAGAATTGCCAAGGAAGAAAAATCCA CCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAATTTGATTTAAAAAGACATCTGGAT 5 GTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATATTTTATATAGAAAAGCTACAAAGT AAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTTTGTTGTATGTATTACAGTCTTTAT AATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATAGACTCAAAACAGTTATTTTTTTAC AATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCTGCTGGATATATTATATGCAATTAA TAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTTTTGGTTGTCATCGATTTACATTGC 10 CACTAATAAACCATATTGAGAATTTCTAAAA >NM_001350597.2 Homo sapiens tRNA methyltransferase 11 homolog (TRMT11), transcript variant 19, mRNA GGGCAGCTGCAATGGCGCTGTCGTGTACCCTTAACAGGTATCTGCTCCTCATGGCGCAGGAGCATCTGGA 15 GTTCCGCCTGCCGGTGAGTCCGTAGCGCCCTCCGGAACTTCCGACGGAAGAGGACGCTGGAGTGGAGTGG AGTGGGTGGAGGTGATCTGCATAGCCCGAGGAAGCTGGGATTCGGGGGTCTTCGCTGGTCTGCGCGGGTC ACGCGTCCCCAGTCCTCGGGCGGGGAAATAAAGTCTTTGCTTTTGCTTTTTGGAGGTCAGTTTGCCAGCA GTCAAGAAACTTATGGAAAGTCACCATTTTGGATTCTTAGCATTCCCTCTGAAGATATTGCAAGAAATTT GATGAAACGGACAGTGTGTGCCAAGTCTATATTTGAACTATGGGGTCATGGACAATCTCCTGAGGAGCTG 20 TACAGTTCTCTTAAAAACTACCCTGTGGAGAAGATGGTTCCATTTCTACATTCGGACTCTACATATAAAA TAAAGATTCACACTTTTAATAAGACATTGACACAAGAAGAGAAAATCAAGCGAATAGATGCACTTGAATT TCTGCCATTTGAAGGAAAAGTGAATTTAAAGAAACCGCAACATGTATTTTCTGTTTTGGAGGATTATGGT TTAGACCCAAACTGCATCCCTGAGAATCCACATAATATTTATTTTGGTAGATGGATTGCAGATGGACAGA GAGAGCTTATTGAGTCATACAGTGTCAAAAAGAGACACTTTATTGGAAATACAAGTATGGATGCTGGTTT 25 GTCATTCATTATGGCTAACCATGGAAAAGTGAAAGAAAATGATATTGTCTTTGATCCATTTGTTGGAACA GCAGTGTACTTGTAAATTGGGTCTTCTCAAAACTCACTCTAACCAAATGGATAAGGTTCTGTTAAGGAAG CACAGGAAAGCTTTTCTGTTCCCCTTGACAACTAGTTCTTCTAGGTGGCCTGCTGATAGCATGTGCTCAT TTTGGTGCATATGTGTATGGGACAGACATAGACTACAACACAGTTCATGGCTTGGGACTGTTTACTGCTG GAAGAAAGGAACCATATTCTCTTCAACTCTATCTTACTACAAAAATCTTCGTACTTGAAAATATCGAATG 30 AGTGTTTTAAGGCAGTTTTACTTGGAAGTTGACAGAACAAGATAAGTTAGACATCAGTACATTTTACTGT TATGTTGAAATTAAAATAACTATTTACTTGTATACATAAAATTAAGATTTTAAATGAAAGCAAATTTTGA ATAAATGTAGGTTTCAGTGAGTTAAAACATTACTTTTCCTAAATGACAGAAAATAAGAATACTCTGTTTA ATACTTTCTCTCTTCCTTTTTCTTTCCCTTCAGGAAAGGCTACTAGGAAAAACCAGAAGTGGAGAGGACC AGATGAAAACATTAGGGCCAATCTTCGTCAATATGGTTTAGAGAAGTATTACCTTGATGTCCTGGTTTCA 35 GATGCATCTAAACCTTCCTGGAGGAAGGGCACATATTTTGATGCAATCATTACTGATCCTCCATATGGTA TCAGAGAATCTACAAGAAGAACAGGTTCACAGAAGGAGATACCAAAGGGGATAGAAAAATGGGAAAAATG TCCAGAAAGCCATGTTCCTGTTTCCTTGAGTTATCATCTGAGTGATATGTTTCTTGACCTGTTAAACTTC GCAGCTGAGACCCTCGTTTTAGGTGGAAGACTAGTCTATTGGTTACCGGTGTATACGCCAGAATACACTG AAGAGATGGTGCCTTGGCACCCTTGCCTGGAACTCGTTAGCAACTGCGAGCAGAAGCTTTCCAGTCACAC 40 ATCAAGGCGCTTGATCACAATGGAAAAGGTGAAGAAATTTGAGAATCGGGACCAGTATTCACATCTGCTA AGTGATCATTTTCTGCCATACCAAGGTCATAATTCCTTCCGTGAGAAATATTTTAGTGGGGTAACAAAAA 63 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GAATTGCCAAGGAAGAAAAATCCACCCAGGAATGAAAATTAAGATTTTGACAATGAAGAAAGAATAAGAA TTTGATTTAAAAAGACATCTGGATGTGAACTTTCATGTATGATCCAGAAAATAGGTACGGTTTTAAAATA TTTTATATAGAAAAGCTACAAAGTAAATTGAGCAATGCTTTTAAAGTTATCTTTGTTTTATAGACTTTTT TGTTGTATGTATTACAGTCTTTATAATCTTATTTAATGTATATTTGTACTTTCAAGTACTGATGGAGATA 5 GACTCAAAACAGTTATTTTTTTACAATTAATCTACAAAGGGAATTAATATTGTTGACTTTTAAAACATCT GCTGGATATATTATATGCAATTAATAGTAGTTAAGAATTTATTCATTTGGTAGATATGTTTATTTGGTTT TTGGTTGTCATCGATTTACATTGCCACTAATAAACCATATTGAGAATTTCTAAAA By “tRNA methyltransferase 13 homolog polypeptide” or “TRMT13 polypeptide” is 10 meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001380338.1, NP_001380339.1, NP_001380340.1, NP_001380341.1, NP_001380342.1, NP_001380343.1, or NP_061956.2, or a fragment thereof, and having methyltransferase activity. The sequences of exemplary TRMT13 polypeptides are provided below: 15 >NP_001380338.1 tRNA:m(4)X modification enzyme TRM13 homolog isoform 2 [Homo sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEEEDARKRILCPLDPKHTVYED QLAKHLKKCNSREKPKPDFYIQDINAGLRDETEIPEQLVPISSLSEEQLEKLIKKLRKASEALHDALNDP KNGDSATKHLKQQASILGNIENLKLLGPRRCFVEFGAGKGKLSHWVDIALKDAEKVHFILVEKVTTRFKV 20 DGKHRKKNSVFERLQIDIQHLCLNKIPVLREEKLPVVGIGKHLCGMATDLALRCLVETYAASFEERNEEP LAKRIKNDKTEKEIYTLAKEGNEKNVPEKWNPVAGIVIALCCHHRCDWRHYVGKEYFRALGLGAVEFHYF QRMSSWATCGMRKTSLETSNSTTKRQDNQNDDSEEHDDGGYRITDDGADCLPGLLSVEEKKKIGHLCKLL IDQGRIQYLQQKGFSPALQYYTDPLVSLENVLLTALPNHSSSPETTA 25 >NP_001380339.1 tRNA:m(4)X modification enzyme TRM13 homolog isoform 3 [Homo sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEDFYIQDINAGLRDETEIPEQL VPISSLSEEQLEKLIKKLRKASEGLNSTLKDHIMSHPALHDALNDPKNGDSATKHLKQQASILGNIENLK LLGPRRCFVEFGAGKGKLSHWVDIALKDAEKVHFILVEKVTTRFKVDGKHRKKNSVFERLQIDIQHLCLN 30 KIPVLREEKLPVVGIGKHLCGMATDLALRCLVETYAASFEERNEEPLAKRIKNDKTEKEIYTLAKEGNEK NVPEKWNPVAGIVIALCCHHRCDWRHYVGKEYFRALGLGAVEFHYFQRMSSWATCGMRKTSLETSNSTTK RQDNQNDDSEEHDDGGYRITDDGADCLPGLLSVEEKKKIGHLCKLLIDQGRIQYLQQKGFSPALQYYTDP LVSLENVLLTALPNHSSSPETTA 35 >NP_001380340.1 tRNA:m(4)X modification enzyme TRM13 homolog isoform 4 [Homo sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEEEDARKRILCPLDPKHTVYED QLAKHLKKCNSREKPKPDFYIQDINAGLRDETEIPEQLVPISSLSEEQLEKLIKKLRKASEGLNSTLKDH IMSHPALHDALNDPKNGDSATKHLKQQASILGNIENLKLLGPRRCFVEFGAGKGKLSHWVDIALKDAEKV 64 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 HFILVEKVTTRFKVDGKHRKKNSVFERLQIDIQHLCLNKIPVLREEKLPVVGIGKHLCGMATDGVSLCRP DWSAVAQSRLTATSASRLQAILLPQPPELLGLQTRATTPG >NP_001380341.1 tRNA:m(4)X modification enzyme TRM13 homolog isoform 5 [Homo 5 sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEEEDARKRILCPLDPKHTVYED QLAKHLKKCNSREKPKPDFYIQDINAGLRDETEIPEQLVPISSLSEEQLEKLIKKLRKASEALHDALNDP KNGDSATKHLKQQASILGNIENLKLLGPRRCFVEFGAGKGKLSHWVDIALKDAEKVHFILVEKVTTRFKV DGKHRKKNSVFERLQIDIQHLCLNKIPVLREEKLPVVGIGKHLCGMATDGVSLCRPDWSAVAQSRLTATS 10 ASRLQAILLPQPPELLGLQTRATTPG >NP_001380342.1 tRNA:m(4)X modification enzyme TRM13 homolog isoform 6 [Homo sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEEEDARKRILCPLDPKHTVYED 15 QLAKHLKKCNSREKPKPDFYIQDINAGLRDETEIPEQLVPISSLSEEQLEKLIKKLRKASEGLNSTLKDH IMSHPALHDALNDPKNGDSATKHLKQQASILGNIENLKLLGPRRCFVEFGAGKGKLSHWVDIALKDAEKV HFILVEKVTTRFKVDGKHRKKNSVFERLQIDIQHLCLNKIPVLREEKLPVVGIGKHLCGMATGFLVLKKR RK 20 >NP_001380343.1 tRNA:m(4)X modification enzyme TRM13 homolog isoform 7 [Homo sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEEEDARKRILCPLDPKHTVYED QLAKHLKKCNSREKPKPDFYIQDINAGLRDETEIPEQLVPISSLSEEQLEKLIKKLRKASEALHDALNDP KNGDSATKHLKQQASILGNIENLKLLGPRRCFVEFGAGKGKLSHWVDIALKDAEKVHFILVEKVTTRFKV 25 DGKHRKKNSVFERLQIDIQHLCLNKIPVLREEKLPVVGIGKHLCGMATGFLVLKKRRK >NP_061956.2 tRNA:m(4)X modification enzyme TRM13 homolog isoform 1 [Homo sapiens] MATSATSPHAPGFPAEGRCGYYVEKKKRFCRMVVAAGKRFCGEHAGAAEEEDARKRILCPLDPKHTVYED 30 QLAKHLKKCNSREKPKPDFYIQDINAGLRDETEIPEQLVPISSLSEEQLEKLIKKLRKASEGLNSTLKDH IMSHPALHDALNDPKNGDSATKHLKQQASILGNIENLKLLGPRRCFVEFGAGKGKLSHWVDIALKDAEKV HFILVEKVTTRFKVDGKHRKKNSVFERLQIDIQHLCLNKIPVLREEKLPVVGIGKHLCGMATDLALRCLV ETYAASFEERNEEPLAKRIKNDKTEKEIYTLAKEGNEKNVPEKWNPVAGIVIALCCHHRCDWRHYVGKEY FRALGLGAVEFHYFQRMSSWATCGMRKTSLETSNSTTKRQDNQNDDSEEHDDGGYRITDDGADCLPGLLS 35 VEEKKKIGHLCKLLIDQGRIQYLQQKGFSPALQYYTDPLVSLENVLLTALPNHSSSPETTA By “tRNA methyltransferase 13 homolog polynucleotide” or “TRMT13 polynucleotide” is meant a nucleic acid molecule encoding an TRMT13 polypeptide or fragment thereof. The sequences of exemplary TRMT13 polynucleotides are provided below: 65 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 >NM_001393409.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), transcript variant 2, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC 5 TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC AAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAACAATTAGTTCCAATTTCTTCTCTATC TGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAAGTGAAGCATTACACGATGCACTTAAT GACCCTAAAAATGGCGATTCTGCAACCAAGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAA 10 ATTTAAAGTTACTTGGTCCAAGAAGATGCTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTG GGTTGATATTGCCTTAAAAGATGCTGAAAAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTC AAGGTGGATGGAAAACACAGAAAGAAAAATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGT GTTTGAACAAGATTCCTGTGCTAAGAGAAGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGG TATGGCAACAGATCTTGCATTACGATGTTTGGTTGAAACCTATGCTGCCAGTTTTGAGGAAAGGAATGAA 15 GAACCTTTAGCCAAACGCATAAAGAATGATAAAACAGAAAAAGAAATTTACACTTTGGCCAAGGAAGGAA ATGAAAAAAATGTCCCAGAGAAGTGGAACCCTGTGGCTGGCATTGTTATTGCACTCTGTTGTCACCACAG GTGTGATTGGAGACATTATGTGGGCAAAGAATATTTCAGGGCTCTAGGCCTTGGAGCAGTGGAATTCCAT TATTTCCAGCGAATGAGTAGTTGGGCAACTTGTGGGATGCGGAAAACATCTTTGGAAACCTCAAATAGTA CCACAAAGAGGCAAGATAATCAGAATGATGATAGTGAAGAGCATGATGATGGAGGATACAGAATCACAGA 20 TGATGGCGCTGATTGTTTGCCTGGGCTTCTTAGTGTTGAAGAAAAGAAGAAAATAGGGCATCTTTGTAAA TTGCTGATTGACCAAGGTCGAATCCAGTATTTGCAGCAGAAGGGATTCAGTCCTGCTTTGCAGTACTATA CAGACCCTCTGGTGTCTTTGGAAAATGTTTTGTTAACTGCTTTACCAAATCATTCTTCATCACCAGAAAC AACTGCTTAATGGAAAAGAAATTTGAGCATCATCTGTCTTCCACCAAAAAAAATTTTTTAATTATATTTT TATATCAAAAAAATATATACTTTAAATAGCAAATAATATGAACTTTAAAAAATGCTGTGGCCTCATTAAA 25 CTTTGGTAATAGCTTTTCTTTTTACTTCAGAAATCCAAACATTAGAGAATTCACCAAAGTAATCCTCTTT AGAAGGGCATCTTGAATTATATTATCCATCCGTATTTATGGAGATTGGTAAAGTAGTTGAACCGTTGACT TGGTGATCTGAAACATACATAACATGTCAACACATAATTAGCGTATTTCTGTTTGTATTAATTTTGGAAA TTTATCTTCCTTTGATTTTATTTAATATTTTTTATTTCTTAAGTTCAAGAGGTAGTGATTGATTGTAACA AGGGCCAATCTGAGAATTTGACTTATATGTGGACATTTTTCCCTACAGATCTGGAAAGCACAATTGTGAT 30 TTTCTCAAATGCAGACAATTCAGAGATATTCACAATTAATAAACACAATTAATTAATGAAGTCACCTTCA AATTTCCAGAGCCATACATGTATATATTGTCAGAATCTGTCCATGACAAACACAAAACTGAAGAGCTGTT TTCAAAGAAAAAAGATTATTTCACTTAATTATTTTGTTGGATAATTGTCTAGTTAGAACTTCCAAAAAGA TACTTACAAACATAATTCACAAATTTGAAATAATTTCTGAAGTTGATTAGCTATCTCATATCTTTTATCA GGTCATTTTTTATATATGTAGGCACAAACAATAAGTATGTTCTCTTCTGTTTGGGAAAATAATTTGGAAT 35 AAAATAGAAATTAGATAATGAAACCAAAAACCTTCTCAAATTTAGGCCTTATTTAACTCATGACTGGTTT CTATGCACAAGAAAATACTAAACCAAATATATGGTAGGACTGTTATTCTCTTCTGCATCTTATCCCTATT TTGTTTCTGCCTTTATTTGTAAAAATTGTCTTAATTGATGGTACATTTGCCAAGACAAAGGTTCAGAATT ACTAATTTTAGATATTATGATATTCTGAAATAACTATTTTTATCCTGTAGTTCTATGATTATATGATTTG TAAATAAGAAGCCTAACCAATTTAAAATTCCTGACTTTAGTCTCATATATCTTGTCAGACTGTCTCGGTT 40 CAAATTCCAAACCTACCATCTTCAGTTGTGCGACCTTGGGCATGCTACCTAATCTCTTTGTGCCTCAATT TCCTCCTTTTAAAATGGGGATGATGATGATAACAATAATACCTACCTCACAAGGTTGTTGTGAGCATCAA 66 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ATGAGATAACACAAGTAAAATGCATAGAACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAGTA ATAGTAGTGGTAGTAACTCGTGAATCTTTTTAATAACATAATAGGCTTTGATTTTATTATCTCTTTAAGT TGTTAACTTTTTTCCCTTGTTATAAGTTTTATGTCAAGTAAGGTAGTTTGTTTAAGTTAGTTACCCATGT CCCCAATCAAGGGAACCTAAATGAAGATATATCATTAAATAATTAACCTTTTTTTATTGTGTCAAGCAAT 5 ACTAATTTGTGTCATAAAATTTGTTTTGCATCATTCAACAATTTATGCCCAGAAATAAACAATATTCTAC AACAGG >NM_001393410.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), transcript variant 3, mRNA 10 AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGATTTCTATATTCAAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAA CAATTAGTTCCAATTTCTTCTCTATCTGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAA GTGAAGGCTTGAATTCTACACTTAAAGATCATATTATGTCCCATCCAGCATTACACGATGCACTTAATGA 15 CCCTAAAAATGGCGATTCTGCAACCAAGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAAAT TTAAAGTTACTTGGTCCAAGAAGATGCTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTGGG TTGATATTGCCTTAAAAGATGCTGAAAAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTCAA GGTGGATGGAAAACACAGAAAGAAAAATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGTGT TTGAACAAGATTCCTGTGCTAAGAGAAGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGGTA 20 TGGCAACAGATCTTGCATTACGATGTTTGGTTGAAACCTATGCTGCCAGTTTTGAGGAAAGGAATGAAGA ACCTTTAGCCAAACGCATAAAGAATGATAAAACAGAAAAAGAAATTTACACTTTGGCCAAGGAAGGAAAT GAAAAAAATGTCCCAGAGAAGTGGAACCCTGTGGCTGGCATTGTTATTGCACTCTGTTGTCACCACAGGT GTGATTGGAGACATTATGTGGGCAAAGAATATTTCAGGGCTCTAGGCCTTGGAGCAGTGGAATTCCATTA TTTCCAGCGAATGAGTAGTTGGGCAACTTGTGGGATGCGGAAAACATCTTTGGAAACCTCAAATAGTACC 25 ACAAAGAGGCAAGATAATCAGAATGATGATAGTGAAGAGCATGATGATGGAGGATACAGAATCACAGATG ATGGCGCTGATTGTTTGCCTGGGCTTCTTAGTGTTGAAGAAAAGAAGAAAATAGGGCATCTTTGTAAATT GCTGATTGACCAAGGTCGAATCCAGTATTTGCAGCAGAAGGGATTCAGTCCTGCTTTGCAGTACTATACA GACCCTCTGGTGTCTTTGGAAAATGTTTTGTTAACTGCTTTACCAAATCATTCTTCATCACCAGAAACAA CTGCTTAATGGAAAAGAAATTTGAGCATCATCTGTCTTCCACCAAAAAAAATTTTTTAATTATATTTTTA 30 TATCAAAAAAATATATACTTTAAATAGCAAATAATATGAACTTTAAAAAATGCTGTGGCCTCATTAAACT TTGGTAATAGCTTTTCTTTTTACTTCAGAAATCCAAACATTAGAGAATTCACCAAAGTAATCCTCTTTAG AAGGGCATCTTGAATTATATTATCCATCCGTATTTATGGAGATTGGTAAAGTAGTTGAACCGTTGACTTG GTGATCTGAAACATACATAACATGTCAACACATAATTAGCGTATTTCTGTTTGTATTAATTTTGGAAATT TATCTTCCTTTGATTTTATTTAATATTTTTTATTTCTTAAGTTCAAGAGGTAGTGATTGATTGTAACAAG 35 GGCCAATCTGAGAATTTGACTTATATGTGGACATTTTTCCCTACAGATCTGGAAAGCACAATTGTGATTT TCTCAAATGCAGACAATTCAGAGATATTCACAATTAATAAACACAATTAATTAATGAAGTCACCTTCAAA TTTCCAGAGCCATACATGTATATATTGTCAGAATCTGTCCATGACAAACACAAAACTGAAGAGCTGTTTT CAAAGAAAAAAGATTATTTCACTTAATTATTTTGTTGGATAATTGTCTAGTTAGAACTTCCAAAAAGATA CTTACAAACATAATTCACAAATTTGAAATAATTTCTGAAGTTGATTAGCTATCTCATATCTTTTATCAGG 40 TCATTTTTTATATATGTAGGCACAAACAATAAGTATGTTCTCTTCTGTTTGGGAAAATAATTTGGAATAA AATAGAAATTAGATAATGAAACCAAAAACCTTCTCAAATTTAGGCCTTATTTAACTCATGACTGGTTTCT 67 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ATGCACAAGAAAATACTAAACCAAATATATGGTAGGACTGTTATTCTCTTCTGCATCTTATCCCTATTTT GTTTCTGCCTTTATTTGTAAAAATTGTCTTAATTGATGGTACATTTGCCAAGACAAAGGTTCAGAATTAC TAATTTTAGATATTATGATATTCTGAAATAACTATTTTTATCCTGTAGTTCTATGATTATATGATTTGTA AATAAGAAGCCTAACCAATTTAAAATTCCTGACTTTAGTCTCATATATCTTGTCAGACTGTCTCGGTTCA 5 AATTCCAAACCTACCATCTTCAGTTGTGCGACCTTGGGCATGCTACCTAATCTCTTTGTGCCTCAATTTC CTCCTTTTAAAATGGGGATGATGATGATAACAATAATACCTACCTCACAAGGTTGTTGTGAGCATCAAAT GAGATAACACAAGTAAAATGCATAGAACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAGTAAT AGTAGTGGTAGTAACTCGTGAATCTTTTTAATAACATAATAGGCTTTGATTTTATTATCTCTTTAAGTTG TTAACTTTTTTCCCTTGTTATAAGTTTTATGTCAAGTAAGGTAGTTTGTTTAAGTTAGTTACCCATGTCC 10 CCAATCAAGGGAACCTAAATGAAGATATATCATTAAATAATTAACCTTTTTTTATTGTGTCAAGCAATAC TAATTTGTGTCATAAAATTTGTTTTGCATCATTCAACAATTTATGCCCAGAAATAAACAATATTCTACAA CAGG >NM_001393411.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), 15 transcript variant 4, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC 20 AAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAACAATTAGTTCCAATTTCTTCTCTATC TGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAAGTGAAGGCTTGAATTCTACACTTAAA GATCATATTATGTCCCATCCAGCATTACACGATGCACTTAATGACCCTAAAAATGGCGATTCTGCAACCA AGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAAATTTAAAGTTACTTGGTCCAAGAAGATG CTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTGGGTTGATATTGCCTTAAAAGATGCTGAA 25 AAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTCAAGGTGGATGGAAAACACAGAAAGAAAA ATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGTGTTTGAACAAGATTCCTGTGCTAAGAGA AGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGGTATGGCAACAGACGGAGTCTCGCTCTGT CGCCCAGACTGGAGTGCAGTGGCGCAATCTCGGCTCACTGCAACCTCCGCCTCCCGGCTTCAAGCGATTC TCCTGCCTCAGCCTCCCGAATTGCTGGGACTACAGACTCGTGCCACCACGCCCGGCTAATTTTTGTATTT 30 TTAGTAGAGACGGGATTTCACCGTGTTAGCCAGGAAGGTCTCCATCTCCTGACCTCGTGATCTGCCCACC TCAGCCGGCGTGAGCTACTGCGCCTGGCCTGAAATAACTTAATTCTTTTAATAATTTATCTGGTTATTCA CATTTCTGAAAATCACTTACTTAAGTCAAATATGAATAAAACAATGACTAAATGTATTTTGTCAGAAAAT AATAGTTTATGAAATAATAGATTAGCTATCACTTTCTGCAAATAATGGGTTTCTAATGTTTGTTGACTTA AATTCATATATTGAATTAAATTTACTAGTGAAATATCAGATCATTTAATAGTCATTTGTGATATCTTAGG 35 GTAATAAAATTTTAAAATATGTATATGTTTTTGATTGAACAGACACAACATGACAATCTAATGATTAAGA AGATATGGGTCCTACATTTTGGGTCTTTCCAGTATTGTCAACAGTGTAGGAGTTTTCGATGGGAAATAGC CATAGTCATCAAAAATCCATTTATTTAGTTTTAAAATATACTAAAATATTTTCATTGTAATGTGGAGTAC ATCTGCCTCTTAAGTTTGTGTATATTAATAATTGTATCCTCAGTGCTTTGCACATGAAAGGCATTCAGTA AATGTTTGTTAAATAAATGAATAAATACAGTAAGGCTACTCAGTGAGGCCTGCTTTCCAGATTCTGTTCT 40 GACGCCCTCTTTTCAACTCTTCAGTTCATCACTATTTGCTATGAGCCTCTTCAAGACAAGCATCTTGACC TAGCATCAACCTAAACAGATTTGTCTGCCCTTCCTCCCTCCCTTCCTTCCTTCCTTCATTTTTCCTTTCT 68 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TAAACTAGAATGTAAGTGTAGGTGAATAAAGTTTTCCCATACAAGTCATTTCCTCAGCTAAGGTAGGAAA AGTGAGAGACAATTCAGGTAGTTGGGTAAAAGTTCTTCAAATTGTAGATCTTAAGCTTCAGATAGCTTTA TTGGGAAGTAGGCTTTATGCAGAATAAATCATTAAAGAAATTGAGAAGATAGAAGAGCAAATTTGAATGA AGTTGATAATTCATGATAGTATTATTACAAATATAAAGCTGAGTAGGTTTTTGCTGTTTTTATTGTTGAG 5 TTTTTAAAAACAGTACTGCTTAATAAATGTAAAAAGTATTTATTTTTAAAGATGATGTGGTTTGGGGGGG CCACATAATTGTGTTTGCAGATCTTGCATTACGATGTTTGGTTGAAACCTATGCTGCCAGTTTTGAGGAA AGGAATGAAGAACCTTTAGCCAAACGCATAAAGAATGATAAAACAGAAAAAGAAATTTACACTTTGGCCA AGGAAGGAAATGAAAAAAATGTCCCAGAGAAGTGGAACCCTGTGGCTGGCATTGTTATTGCACTCTGTTG TCACCACAGGTGTGATTGGAGACATTATGTGGGCAAAGAATATTTCAGGGCTCTAGGCCTTGGAGCAGTG 10 GAATTCCATTATTTCCAGCGAATGAGTAGTTGGGCAACTTGTGGGATGCGGAAAACATCTTTGGAAACCT CAAATAGTACCACAAAGAGGCAAGATAATCAGAATGATGATAGTGAAGAGCATGATGATGGAGGATACAG AATCACAGATGATGGCGCTGATTGTTTGCCTGGGTAAGAGACTACTTTTGTAATGCATGATACTAAAGGA GAAATATTATATTGTACTGTACTTTAGATGACTATTATCAACAATTCATGAAAATGTATTTTATAATCTA ATTTTAGAATAAGCTAAAATATACACATCTTTTATTGTGGTCATAAATATAATGTGTCTTGGAAGCATGG 15 GACAAAGTACTTAGTACATTATGGGTTAGTTAACAGTCTCTCAATAAATAGTGACAAAAATAATTTTTTA TAAACTTTTGCAAAATTTTAACAATGTTTTGTGTGTGTTTATTCAATTTAGGCTTCTTAGTGTTGAAGAA AAGAAGAAAATAGGGCATCTTTGTAAATTGCTGATTGACCAAGGTCGAATCCAGTATTTGCAGCAGAAGG GATTCAGTCCTGCTTTGCAGTACTATACAGACCCTCTGGTGTCTTTGGAAAATGTTTTGTTAACTGCTTT ACCAAATCATTCTTCATCACCAGAAACAACTGCTTAATGGAAAAGAAATTTGAGCATCATCTGTCTTCCA 20 CCAAAAAAAATTTTTTAATTATATTTTTATATCAAAAAAATATATACTTTAAATAGCAAATAATATGAAC TTTAAAAAATGCTGTGGCCTCATTAAACTTTGGTAATAGCTTTTCTTTTTACTTCAGAAATCCAAACATT AGAGAATTCACCAAAGTAATCCTCTTTAGAAGGGCATCTTGAATTATATTATCCATCCGTATTTATGGAG ATTGGTAAAGTAGTTGAACCGTTGACTTGGTGATCTGAAACATACATAACATGTCAACACATAATTAGCG TATTTCTGTTTGTATTAATTTTGGAAATTTATCTTCCTTTGATTTTATTTAATATTTTTTATTTCTTAAG 25 TTCAAGAGGTAGTGATTGATTGTAACAAGGGCCAATCTGAGAATTTGACTTATATGTGGACATTTTTCCC TACAGATCTGGAAAGCACAATTGTGATTTTCTCAAATGCAGACAATTCAGAGATATTCACAATTAATAAA CACAATTAATTAATGAAGTCACCTTCAAATTTCCAGAGCCATACATGTATATATTGTCAGAATCTGTCCA TGACAAACACAAAACTGAAGAGCTGTTTTCAAAGAAAAAAGATTATTTCACTTAATTATTTTGTTGGATA ATTGTCTAGTTAGAACTTCCAAAAAGATACTTACAAACATAATTCACAAATTTGAAATAATTTCTGAAGT 30 TGATTAGCTATCTCATATCTTTTATCAGGTCATTTTTTATATATGTAGGCACAAACAATAAGTATGTTCT CTTCTGTTTGGGAAAATAATTTGGAATAAAATAGAAATTAGATAATGAAACCAAAAACCTTCTCAAATTT AGGCCTTATTTAACTCATGACTGGTTTCTATGCACAAGAAAATACTAAACCAAATATATGGTAGGACTGT TATTCTCTTCTGCATCTTATCCCTATTTTGTTTCTGCCTTTATTTGTAAAAATTGTCTTAATTGATGGTA CATTTGCCAAGACAAAGGTTCAGAATTACTAATTTTAGATATTATGATATTCTGAAATAACTATTTTTAT 35 CCTGTAGTTCTATGATTATATGATTTGTAAATAAGAAGCCTAACCAATTTAAAATTCCTGACTTTAGTCT CATATATCTTGTCAGACTGTCTCGGTTCAAATTCCAAACCTACCATCTTCAGTTGTGCGACCTTGGGCAT GCTACCTAATCTCTTTGTGCCTCAATTTCCTCCTTTTAAAATGGGGATGATGATGATAACAATAATACCT ACCTCACAAGGTTGTTGTGAGCATCAAATGAGATAACACAAGTAAAATGCATAGAACAGTTCCAAGCACA GAGTAATTCAATAAATATTAACTAGTAATAGTAGTGGTAGTAACTCGTGAATCTTTTTAATAACATAATA 40 GGCTTTGATTTTATTATCTCTTTAAGTTGTTAACTTTTTTCCCTTGTTATAAGTTTTATGTCAAGTAAGG TAGTTTGTTTAAGTTAGTTACCCATGTCCCCAATCAAGGGAACCTAAATGAAGATATATCATTAAATAAT 69 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TAACCTTTTTTTATTGTGTCAAGCAATACTAATTTGTGTCATAAAATTTGTTTTGCATCATTCAACAATT TATGCCCAGAAATAAACAATATTCTACAACAGG >NM_001393412.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), 5 transcript variant 5, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC 10 AAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAACAATTAGTTCCAATTTCTTCTCTATC TGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAAGTGAAGCATTACACGATGCACTTAAT GACCCTAAAAATGGCGATTCTGCAACCAAGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAA ATTTAAAGTTACTTGGTCCAAGAAGATGCTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTG GGTTGATATTGCCTTAAAAGATGCTGAAAAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTC 15 AAGGTGGATGGAAAACACAGAAAGAAAAATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGT GTTTGAACAAGATTCCTGTGCTAAGAGAAGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGG TATGGCAACAGACGGAGTCTCGCTCTGTCGCCCAGACTGGAGTGCAGTGGCGCAATCTCGGCTCACTGCA ACCTCCGCCTCCCGGCTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAATTGCTGGGACTACAGACTCGTG CCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGACGGGATTTCACCGTGTTAGCCAGGAAGGTCTC 20 CATCTCCTGACCTCGTGATCTGCCCACCTCAGCCGGCGTGAGCTACTGCGCCTGGCCTGAAATAACTTAA TTCTTTTAATAATTTATCTGGTTATTCACATTTCTGAAAATCACTTACTTAAGTCAAATATGAATAAAAC AATGACTAAATGTATTTTGTCAGAAAATAATAGTTTATGAAATAATAGATTAGCTATCACTTTCTGCAAA TAATGGGTTTCTAATGTTTGTTGACTTAAATTCATATATTGAATTAAATTTACTAGTGAAATATCAGATC ATTTAATAGTCATTTGTGATATCTTAGGGTAATAAAATTTTAAAATATGTATATGTTTTTGATTGAACAG 25 ACACAACATGACAATCTAATGATTAAGAAGATATGGGTCCTACATTTTGGGTCTTTCCAGTATTGTCAAC AGTGTAGGAGTTTTCGATGGGAAATAGCCATAGTCATCAAAAATCCATTTATTTAGTTTTAAAATATACT AAAATATTTTCATTGTAATGTGGAGTACATCTGCCTCTTAAGTTTGTGTATATTAATAATTGTATCCTCA GTGCTTTGCACATGAAAGGCATTCAGTAAATGTTTGTTAAATAAATGAATAAATACAGTAAGGCTACTCA GTGAGGCCTGCTTTCCAGATTCTGTTCTGACGCCCTCTTTTCAACTCTTCAGTTCATCACTATTTGCTAT 30 GAGCCTCTTCAAGACAAGCATCTTGACCTAGCATCAACCTAAACAGATTTGTCTGCCCTTCCTCCCTCCC TTCCTTCCTTCCTTCATTTTTCCTTTCTTAAACTAGAATGTAAGTGTAGGTGAATAAAGTTTTCCCATAC AAGTCATTTCCTCAGCTAAGGTAGGAAAAGTGAGAGACAATTCAGGTAGTTGGGTAAAAGTTCTTCAAAT TGTAGATCTTAAGCTTCAGATAGCTTTATTGGGAAGTAGGCTTTATGCAGAATAAATCATTAAAGAAATT GAGAAGATAGAAGAGCAAATTTGAATGAAGTTGATAATTCATGATAGTATTATTACAAATATAAAGCTGA 35 GTAGGTTTTTGCTGTTTTTATTGTTGAGTTTTTAAAAACAGTACTGCTTAATAAATGTAAAAAGTATTTA TTTTTAAAGATGATGTGGTTTGGGGGGGCCACATAATTGTGTTTGCAGATCTTGCATTACGATGTTTGGT TGAAACCTATGCTGCCAGTTTTGAGGAAAGGAATGAAGAACCTTTAGCCAAACGCATAAAGAATGATAAA ACAGAAAAAGAAATTTACACTTTGGCCAAGGAAGGAAATGAAAAAAATGTCCCAGAGAAGTGGAACCCTG TGGCTGGCATTGTTATTGCACTCTGTTGTCACCACAGGTGTGATTGGAGACATTATGTGGGCAAAGAATA 40 TTTCAGGGCTCTAGGCCTTGGAGCAGTGGAATTCCATTATTTCCAGCGAATGAGTAGTTGGGCAACTTGT GGGATGCGGAAAACATCTTTGGAAACCTCAAATAGTACCACAAAGAGGCAAGATAATCAGAATGATGATA 70 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GTGAAGAGCATGATGATGGAGGATACAGAATCACAGATGATGGCGCTGATTGTTTGCCTGGGTAAGAGAC TACTTTTGTAATGCATGATACTAAAGGAGAAATATTATATTGTACTGTACTTTAGATGACTATTATCAAC AATTCATGAAAATGTATTTTATAATCTAATTTTAGAATAAGCTAAAATATACACATCTTTTATTGTGGTC ATAAATATAATGTGTCTTGGAAGCATGGGACAAAGTACTTAGTACATTATGGGTTAGTTAACAGTCTCTC 5 AATAAATAGTGACAAAAATAATTTTTTATAAACTTTTGCAAAATTTTAACAATGTTTTGTGTGTGTTTAT TCAATTTAGGCTTCTTAGTGTTGAAGAAAAGAAGAAAATAGGGCATCTTTGTAAATTGCTGATTGACCAA GGTCGAATCCAGTATTTGCAGCAGAAGGGATTCAGTCCTGCTTTGCAGTACTATACAGACCCTCTGGTGT CTTTGGAAAATGTTTTGTTAACTGCTTTACCAAATCATTCTTCATCACCAGAAACAACTGCTTAATGGAA AAGAAATTTGAGCATCATCTGTCTTCCACCAAAAAAAATTTTTTAATTATATTTTTATATCAAAAAAATA 10 TATACTTTAAATAGCAAATAATATGAACTTTAAAAAATGCTGTGGCCTCATTAAACTTTGGTAATAGCTT TTCTTTTTACTTCAGAAATCCAAACATTAGAGAATTCACCAAAGTAATCCTCTTTAGAAGGGCATCTTGA ATTATATTATCCATCCGTATTTATGGAGATTGGTAAAGTAGTTGAACCGTTGACTTGGTGATCTGAAACA TACATAACATGTCAACACATAATTAGCGTATTTCTGTTTGTATTAATTTTGGAAATTTATCTTCCTTTGA TTTTATTTAATATTTTTTATTTCTTAAGTTCAAGAGGTAGTGATTGATTGTAACAAGGGCCAATCTGAGA 15 ATTTGACTTATATGTGGACATTTTTCCCTACAGATCTGGAAAGCACAATTGTGATTTTCTCAAATGCAGA CAATTCAGAGATATTCACAATTAATAAACACAATTAATTAATGAAGTCACCTTCAAATTTCCAGAGCCAT ACATGTATATATTGTCAGAATCTGTCCATGACAAACACAAAACTGAAGAGCTGTTTTCAAAGAAAAAAGA TTATTTCACTTAATTATTTTGTTGGATAATTGTCTAGTTAGAACTTCCAAAAAGATACTTACAAACATAA TTCACAAATTTGAAATAATTTCTGAAGTTGATTAGCTATCTCATATCTTTTATCAGGTCATTTTTTATAT 20 ATGTAGGCACAAACAATAAGTATGTTCTCTTCTGTTTGGGAAAATAATTTGGAATAAAATAGAAATTAGA TAATGAAACCAAAAACCTTCTCAAATTTAGGCCTTATTTAACTCATGACTGGTTTCTATGCACAAGAAAA TACTAAACCAAATATATGGTAGGACTGTTATTCTCTTCTGCATCTTATCCCTATTTTGTTTCTGCCTTTA TTTGTAAAAATTGTCTTAATTGATGGTACATTTGCCAAGACAAAGGTTCAGAATTACTAATTTTAGATAT TATGATATTCTGAAATAACTATTTTTATCCTGTAGTTCTATGATTATATGATTTGTAAATAAGAAGCCTA 25 ACCAATTTAAAATTCCTGACTTTAGTCTCATATATCTTGTCAGACTGTCTCGGTTCAAATTCCAAACCTA CCATCTTCAGTTGTGCGACCTTGGGCATGCTACCTAATCTCTTTGTGCCTCAATTTCCTCCTTTTAAAAT GGGGATGATGATGATAACAATAATACCTACCTCACAAGGTTGTTGTGAGCATCAAATGAGATAACACAAG TAAAATGCATAGAACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAGTAATAGTAGTGGTAGTA ACTCGTGAATCTTTTTAATAACATAATAGGCTTTGATTTTATTATCTCTTTAAGTTGTTAACTTTTTTCC 30 CTTGTTATAAGTTTTATGTCAAGTAAGGTAGTTTGTTTAAGTTAGTTACCCATGTCCCCAATCAAGGGAA CCTAAATGAAGATATATCATTAAATAATTAACCTTTTTTTATTGTGTCAAGCAATACTAATTTGTGTCAT AAAATTTGTTTTGCATCATTCAACAATTTATGCCCAGAAATAAACAATATTCTACAACAGG >NM_001393413.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), 35 transcript variant 6, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC 40 AAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAACAATTAGTTCCAATTTCTTCTCTATC TGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAAGTGAAGGCTTGAATTCTACACTTAAA 71 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 GATCATATTATGTCCCATCCAGCATTACACGATGCACTTAATGACCCTAAAAATGGCGATTCTGCAACCA AGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAAATTTAAAGTTACTTGGTCCAAGAAGATG CTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTGGGTTGATATTGCCTTAAAAGATGCTGAA AAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTCAAGGTGGATGGAAAACACAGAAAGAAAA 5 ATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGTGTTTGAACAAGATTCCTGTGCTAAGAGA AGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGGTATGGCAACAGGCTTCTTAGTGTTGAAG AAAAGAAGAAAATAGGGCATCTTTGTAAATTGCTGATTGACCAAGGTCGAATCCAGTATTTGCAGCAGAA GGGATTCAGTCCTGCTTTGCAGTACTATACAGACCCTCTGGTGTCTTTGGAAAATGTTTTGTTAACTGCT TTACCAAATCATTCTTCATCACCAGAAACAACTGCTTAATGGAAAAGAAATTTGAGCATCATCTGTCTTC 10 CACCAAAAAAAATTTTTTAATTATATTTTTATATCAAAAAAATATATACTTTAAATAGCAAATAATATGA ACTTTAAAAAATGCTGTGGCCTCATTAAACTTTGGTAATAGCTTTTCTTTTTACTTCAGAAATCCAAACA TTAGAGAATTCACCAAAGTAATCCTCTTTAGAAGGGCATCTTGAATTATATTATCCATCCGTATTTATGG AGATTGGTAAAGTAGTTGAACCGTTGACTTGGTGATCTGAAACATACATAACATGTCAACACATAATTAG CGTATTTCTGTTTGTATTAATTTTGGAAATTTATCTTCCTTTGATTTTATTTAATATTTTTTATTTCTTA 15 AGTTCAAGAGGTAGTGATTGATTGTAACAAGGGCCAATCTGAGAATTTGACTTATATGTGGACATTTTTC CCTACAGATCTGGAAAGCACAATTGTGATTTTCTCAAATGCAGACAATTCAGAGATATTCACAATTAATA AACACAATTAATTAATGAAGTCACCTTCAAATTTCCAGAGCCATACATGTATATATTGTCAGAATCTGTC CATGACAAACACAAAACTGAAGAGCTGTTTTCAAAGAAAAAAGATTATTTCACTTAATTATTTTGTTGGA TAATTGTCTAGTTAGAACTTCCAAAAAGATACTTACAAACATAATTCACAAATTTGAAATAATTTCTGAA 20 GTTGATTAGCTATCTCATATCTTTTATCAGGTCATTTTTTATATATGTAGGCACAAACAATAAGTATGTT CTCTTCTGTTTGGGAAAATAATTTGGAATAAAATAGAAATTAGATAATGAAACCAAAAACCTTCTCAAAT TTAGGCCTTATTTAACTCATGACTGGTTTCTATGCACAAGAAAATACTAAACCAAATATATGGTAGGACT GTTATTCTCTTCTGCATCTTATCCCTATTTTGTTTCTGCCTTTATTTGTAAAAATTGTCTTAATTGATGG TACATTTGCCAAGACAAAGGTTCAGAATTACTAATTTTAGATATTATGATATTCTGAAATAACTATTTTT 25 ATCCTGTAGTTCTATGATTATATGATTTGTAAATAAGAAGCCTAACCAATTTAAAATTCCTGACTTTAGT CTCATATATCTTGTCAGACTGTCTCGGTTCAAATTCCAAACCTACCATCTTCAGTTGTGCGACCTTGGGC ATGCTACCTAATCTCTTTGTGCCTCAATTTCCTCCTTTTAAAATGGGGATGATGATGATAACAATAATAC CTACCTCACAAGGTTGTTGTGAGCATCAAATGAGATAACACAAGTAAAATGCATAGAACAGTTCCAAGCA CAGAGTAATTCAATAAATATTAACTAGTAATAGTAGTGGTAGTAACTCGTGAATCTTTTTAATAACATAA 30 TAGGCTTTGATTTTATTATCTCTTTAAGTTGTTAACTTTTTTCCCTTGTTATAAGTTTTATGTCAAGTAA GGTAGTTTGTTTAAGTTAGTTACCCATGTCCCCAATCAAGGGAACCTAAATGAAGATATATCATTAAATA ATTAACCTTTTTTTATTGTGTCAAGCAATACTAATTTGTGTCATAAAATTTGTTTTGCATCATTCAACAA TTTATGCCCAGAAATAAACAATATTCTACAACAGG 35 >NM_001393414.1 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), transcript variant 7, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT 40 GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC AAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAACAATTAGTTCCAATTTCTTCTCTATC 72 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAAGTGAAGCATTACACGATGCACTTAAT GACCCTAAAAATGGCGATTCTGCAACCAAGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAA ATTTAAAGTTACTTGGTCCAAGAAGATGCTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTG GGTTGATATTGCCTTAAAAGATGCTGAAAAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTC 5 AAGGTGGATGGAAAACACAGAAAGAAAAATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGT GTTTGAACAAGATTCCTGTGCTAAGAGAAGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGG TATGGCAACAGGCTTCTTAGTGTTGAAGAAAAGAAGAAAATAGGGCATCTTTGTAAATTGCTGATTGACC AAGGTCGAATCCAGTATTTGCAGCAGAAGGGATTCAGTCCTGCTTTGCAGTACTATACAGACCCTCTGGT GTCTTTGGAAAATGTTTTGTTAACTGCTTTACCAAATCATTCTTCATCACCAGAAACAACTGCTTAATGG 10 AAAAGAAATTTGAGCATCATCTGTCTTCCACCAAAAAAAATTTTTTAATTATATTTTTATATCAAAAAAA TATATACTTTAAATAGCAAATAATATGAACTTTAAAAAATGCTGTGGCCTCATTAAACTTTGGTAATAGC TTTTCTTTTTACTTCAGAAATCCAAACATTAGAGAATTCACCAAAGTAATCCTCTTTAGAAGGGCATCTT GAATTATATTATCCATCCGTATTTATGGAGATTGGTAAAGTAGTTGAACCGTTGACTTGGTGATCTGAAA CATACATAACATGTCAACACATAATTAGCGTATTTCTGTTTGTATTAATTTTGGAAATTTATCTTCCTTT 15 GATTTTATTTAATATTTTTTATTTCTTAAGTTCAAGAGGTAGTGATTGATTGTAACAAGGGCCAATCTGA GAATTTGACTTATATGTGGACATTTTTCCCTACAGATCTGGAAAGCACAATTGTGATTTTCTCAAATGCA GACAATTCAGAGATATTCACAATTAATAAACACAATTAATTAATGAAGTCACCTTCAAATTTCCAGAGCC ATACATGTATATATTGTCAGAATCTGTCCATGACAAACACAAAACTGAAGAGCTGTTTTCAAAGAAAAAA GATTATTTCACTTAATTATTTTGTTGGATAATTGTCTAGTTAGAACTTCCAAAAAGATACTTACAAACAT 20 AATTCACAAATTTGAAATAATTTCTGAAGTTGATTAGCTATCTCATATCTTTTATCAGGTCATTTTTTAT ATATGTAGGCACAAACAATAAGTATGTTCTCTTCTGTTTGGGAAAATAATTTGGAATAAAATAGAAATTA GATAATGAAACCAAAAACCTTCTCAAATTTAGGCCTTATTTAACTCATGACTGGTTTCTATGCACAAGAA AATACTAAACCAAATATATGGTAGGACTGTTATTCTCTTCTGCATCTTATCCCTATTTTGTTTCTGCCTT TATTTGTAAAAATTGTCTTAATTGATGGTACATTTGCCAAGACAAAGGTTCAGAATTACTAATTTTAGAT 25 ATTATGATATTCTGAAATAACTATTTTTATCCTGTAGTTCTATGATTATATGATTTGTAAATAAGAAGCC TAACCAATTTAAAATTCCTGACTTTAGTCTCATATATCTTGTCAGACTGTCTCGGTTCAAATTCCAAACC TACCATCTTCAGTTGTGCGACCTTGGGCATGCTACCTAATCTCTTTGTGCCTCAATTTCCTCCTTTTAAA ATGGGGATGATGATGATAACAATAATACCTACCTCACAAGGTTGTTGTGAGCATCAAATGAGATAACACA AGTAAAATGCATAGAACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAGTAATAGTAGTGGTAG 30 TAACTCGTGAATCTTTTTAATAACATAATAGGCTTTGATTTTATTATCTCTTTAAGTTGTTAACTTTTTT CCCTTGTTATAAGTTTTATGTCAAGTAAGGTAGTTTGTTTAAGTTAGTTACCCATGTCCCCAATCAAGGG AACCTAAATGAAGATATATCATTAAATAATTAACCTTTTTTTATTGTGTCAAGCAATACTAATTTGTGTC ATAAAATTTGTTTTGCATCATTCAACAATTTATGCCCAGAAATAAACAATATTCTACAACAGG 35 >NM_019083.3 Homo sapiens tRNA methyltransferase 13 homolog (TRMT13), transcript variant 1, mRNA AGAATTATGGCGACCTCCGCGACGTCGCCGCACGCGCCTGGTTTTCCAGCTGAGGGTAGATGCGGTTACT ATGTGGAAAAGAAGAAACGGTTCTGCAGGATGGTGGTGGCCGCAGGGAAAAGATTTTGTGGTGAACACGC TGGAGCCGCGGAGGAAGAAGATGCTCGGAAAAGAATCCTGTGTCCTTTAGATCCAAAACACACAGTATAT 40 GAAGATCAACTAGCAAAGCATTTGAAAAAATGTAACTCAAGAGAGAAACCAAAACCTGATTTCTATATTC AAGATATTAATGCAGGCTTAAGAGATGAAACAGAAATACCTGAACAATTAGTTCCAATTTCTTCTCTATC 73 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 TGAAGAGCAGTTGGAAAAGTTAATTAAGAAATTGAGAAAAGCAAGTGAAGGCTTGAATTCTACACTTAAA GATCATATTATGTCCCATCCAGCATTACACGATGCACTTAATGACCCTAAAAATGGCGATTCTGCAACCA AGCACCTGAAACAGCAGGCTTCTATTTTAGGTAACATTGAAAATTTAAAGTTACTTGGTCCAAGAAGATG CTTTGTTGAGTTTGGAGCGGGAAAGGGAAAATTATCTCATTGGGTTGATATTGCCTTAAAAGATGCTGAA 5 AAAGTTCACTTCATCCTAGTGGAAAAGGTGACCACAAGATTCAAGGTGGATGGAAAACACAGAAAGAAAA ATTCAGTGTTTGAAAGACTTCAAATTGATATTCAACACTTGTGTTTGAACAAGATTCCTGTGCTAAGAGA AGAAAAACTACCTGTGGTAGGAATTGGAAAGCATCTGTGTGGTATGGCAACAGATCTTGCATTACGATGT TTGGTTGAAACCTATGCTGCCAGTTTTGAGGAAAGGAATGAAGAACCTTTAGCCAAACGCATAAAGAATG ATAAAACAGAAAAAGAAATTTACACTTTGGCCAAGGAAGGAAATGAAAAAAATGTCCCAGAGAAGTGGAA 10 CCCTGTGGCTGGCATTGTTATTGCACTCTGTTGTCACCACAGGTGTGATTGGAGACATTATGTGGGCAAA GAATATTTCAGGGCTCTAGGCCTTGGAGCAGTGGAATTCCATTATTTCCAGCGAATGAGTAGTTGGGCAA CTTGTGGGATGCGGAAAACATCTTTGGAAACCTCAAATAGTACCACAAAGAGGCAAGATAATCAGAATGA TGATAGTGAAGAGCATGATGATGGAGGATACAGAATCACAGATGATGGCGCTGATTGTTTGCCTGGGCTT CTTAGTGTTGAAGAAAAGAAGAAAATAGGGCATCTTTGTAAATTGCTGATTGACCAAGGTCGAATCCAGT 15 ATTTGCAGCAGAAGGGATTCAGTCCTGCTTTGCAGTACTATACAGACCCTCTGGTGTCTTTGGAAAATGT TTTGTTAACTGCTTTACCAAATCATTCTTCATCACCAGAAACAACTGCTTAATGGAAAAGAAATTTGAGC ATCATCTGTCTTCCACCAAAAAAAATTTTTTAATTATATTTTTATATCAAAAAAATATATACTTTAAATA GCAAATAATATGAACTTTAAAAAATGCTGTGGCCTCATTAAACTTTGGTAATAGCTTTTCTTTTTACTTC AGAAATCCAAACATTAGAGAATTCACCAAAGTAATCCTCTTTAGAAGGGCATCTTGAATTATATTATCCA 20 TCCGTATTTATGGAGATTGGTAAAGTAGTTGAACCGTTGACTTGGTGATCTGAAACATACATAACATGTC AACACATAATTAGCGTATTTCTGTTTGTATTAATTTTGGAAATTTATCTTCCTTTGATTTTATTTAATAT TTTTTATTTCTTAAGTTCAAGAGGTAGTGATTGATTGTAACAAGGGCCAATCTGAGAATTTGACTTATAT GTGGACATTTTTCCCTACAGATCTGGAAAGCACAATTGTGATTTTCTCAAATGCAGACAATTCAGAGATA TTCACAATTAATAAACACAATTAATTAATGAAGTCACCTTCAAATTTCCAGAGCCATACATGTATATATT 25 GTCAGAATCTGTCCATGACAAACACAAAACTGAAGAGCTGTTTTCAAAGAAAAAAGATTATTTCACTTAA TTATTTTGTTGGATAATTGTCTAGTTAGAACTTCCAAAAAGATACTTACAAACATAATTCACAAATTTGA AATAATTTCTGAAGTTGATTAGCTATCTCATATCTTTTATCAGGTCATTTTTTATATATGTAGGCACAAA CAATAAGTATGTTCTCTTCTGTTTGGGAAAATAATTTGGAATAAAATAGAAATTAGATAATGAAACCAAA AACCTTCTCAAATTTAGGCCTTATTTAACTCATGACTGGTTTCTATGCACAAGAAAATACTAAACCAAAT 30 ATATGGTAGGACTGTTATTCTCTTCTGCATCTTATCCCTATTTTGTTTCTGCCTTTATTTGTAAAAATTG TCTTAATTGATGGTACATTTGCCAAGACAAAGGTTCAGAATTACTAATTTTAGATATTATGATATTCTGA AATAACTATTTTTATCCTGTAGTTCTATGATTATATGATTTGTAAATAAGAAGCCTAACCAATTTAAAAT TCCTGACTTTAGTCTCATATATCTTGTCAGACTGTCTCGGTTCAAATTCCAAACCTACCATCTTCAGTTG TGCGACCTTGGGCATGCTACCTAATCTCTTTGTGCCTCAATTTCCTCCTTTTAAAATGGGGATGATGATG 35 ATAACAATAATACCTACCTCACAAGGTTGTTGTGAGCATCAAATGAGATAACACAAGTAAAATGCATAGA ACAGTTCCAAGCACAGAGTAATTCAATAAATATTAACTAGTAATAGTAGTGGTAGTAACTCGTGAATCTT TTTAATAACATAATAGGCTTTGATTTTATTATCTCTTTAAGTTGTTAACTTTTTTCCCTTGTTATAAGTT TTATGTCAAGTAAGGTAGTTTGTTTAAGTTAGTTACCCATGTCCCCAATCAAGGGAACCTAAATGAAGAT ATATCATTAAATAATTAACCTTTTTTTATTGTGTCAAGCAATACTAATTTGTGTCATAAAATTTGTTTTG 40 CATCATTCAACAATTTATGCCCAGAAATAAACAATATTCTACAACAGG 74 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 By “tRNA methyltransferase 44 homolog polypeptide” or “TRMT44 polypeptide” is meant a polypeptide having about 85% or greater amino acid sequence identity to NCBI Accession Nos. NP_001337162.1 or NP_689757.2, or a fragment thereof, and having methyltransferase activity. The sequences of exemplary TRMT44 polypeptides are provided 5 below: >NP_001337162.1 probable tRNA (uracil-O(2)-)-methyltransferase isoform 3 [Homo sapiens] MCIKFSSVIAKKNERWHSDGIVYPKPTWLGEELLAKLAKWSVENKKSDFKSTLSLISIMKYSKAYQELKE KYKEMVKVWPEVTDPEKFVYEDVAIAAYLLILWEEERAERRLTARQSFVDLGCGNGLLVHILSSEGHPGR 10 GIDVRRRKIWDMYGPQTQLEEDAITPNDKTLFPDVDWLIGNHSDELTPWIPVIAARSSYNCRFFVLPCCF FDFIGRYSRRQSKKTQYREYLDFIKEVGFTCGFHVDEDCLRIPSTKRVCLVGKSRTYPSSREASVDEKRT QYIKSRRGCPVSPPGWELSPSPRWVAAGSAGHCDGQQALDARVGCVTRAWAAEHGAGPQAEGPWLPGFHP REKAERVRNCAALPRDFIDQVVLQVANLLLGGKQLNTRSSRNGSLKTWNGGESLSLAEVANELDTETLRR LKRECGGLQTLLRNSHQVFQVVNGRVHIRDWREETLWKTKQPEAKQRLLSEACKTRLCWFFMHHPDGCAL 15 STDCCPFAHGPAELRPPRTTPRKKIS >NP_689757.2 probable tRNA (uracil-O(2)-)-methyltransferase isoform 2 [Homo sapiens] MAEVGRTGISYPGALLPQGFWAAVEVWLERPQVANKRLCGARLEARWSAALPCAEARGPGTSAGSEQKER 20 GPGPGQGSPGGGPGPRSLSGPEQGTACCELEEAQGQCQQEEAQREAASVPLRDSGHPGHAEGREGDFPAA DLDSLWEDFSQSLARGNSELLAFLTSSGAGSQPEAQRELDVVLRTVIPKTSPHCPLTTPRREIVVQDVLN GTITFLPLEEDDEGNLKVKMSNVYQIQLSHSKEEWFISVLIFCPERWHSDGIVYPKPTWLGEELLAKLAK WSVENKKSDFKSTLSLISIMKYSKAYQELKEKYKEMVKVWPEVTDPEKFVYEDVAIAAYLLILWEEERAE RRLTARQSFVDLGCGNGLLVHILSSEGHPGRGIDVRRRKIWDMYGPQTQLEEDAITPNDKTLFPDVDWLI 25 GNHSDELTPWIPVIAARSSYNCRFFVLPCCFFDFIGRYSRRQSKKTQYREYLDFIKEVGFTCGFHVDEDC LRIPSTKRVCLVGKSRTYPSSREASVDEKRTQYIKSRRGCPVSPPGWELSPSPRWVAAGSAGHCDGQQAL DARVGCVTRAWAAEHGAGPQAEGPWLPGFHPREKAERVRNCAALPRDFIDQVVLQVANLLLGGKQLNTRS SRNGSLKTWNGGESLSLAEVANELDTETLRRLKRECGGLQTLLRNSHQVFQVVNGRVHIRDWREETLWKT KQPEAKQRLLSEACKTRLCWFFMHHPDGCALSTDCCPFAHGPAELRPPRTTPRKKIS 30 By “tRNA methyltransferase 44 homolog polynucleotide” or “TRMT44 polynucleotide” is meant a nucleic acid molecule encoding an TRMT44 polypeptide or fragment thereof. The sequences of exemplary TRMT44 polynucleotides are provided below: >NM_001350233.2 Homo sapiens tRNA methyltransferase 44 homolog (TRMT44), 35 transcript variant 3, mRNA GTCATCTCGGCGCGCCGCTGCCAGGGCTGTACACCTGCTGGCTGCCATGGCTGAGGTGGGCCGTACCGGG ATCAGCTACCCAGGCGCGCTTCTCCCACAGGGCTTCTGGGCTGCGGTCGAAGTGTGGCTGGAGAGGCCGC AGGTGGCAAACAAACGGCTTTGCGGCGCCCGCCTGGAGGCCCGCTGGAGCGCCGCCCTGCCCTGCGCGGA GGCCCGCGGCCCCGGGACTAGCGCAGGCTCGGAGCAGAAGGAGCGGGGTCCGGGACCCGGCCAGGGTTCC 75 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 CCCGGAGGGGGCCCGGGTCCCAGGTCGCTATCAGGACCCGAGCAGGGCACGGCATGTTGCGAACTTGAGG AGGCCCAGGGCCAGTGCCAGCAAGAGGAGGCACAGAGGGAAGCCGCCTCAGTGCCCCTGAGGGACTCCGG GCACCCCGGCCATGCTGAAGGAAGGGAGGGCGACTTCCCCGCCGCAGATCTGGATTCGCTTTGGGAGGAT TTCTCCCAAAGTCTCGCCCGTGGCAATTCGGAGTTGCTGGCCTTCCTCACCAGCTCCGGGGCGGGATCGC 5 AGCCAGAGGCGCAGCGTGAGCTCGACGTGGTTCTCAGAACCGTCATCCCGAAAACTAGCCCACATTGCCC CCTTACAACTCCCAGGAGGGAAATAGTCGTGCAAGATGTCCTCAATGGAACCATAACGTTTTTGCCTTTG GAAGAAGATGATGAGGGGAACCTAAAGGTTAAGATGAGCAATGTGTATCAAATTCAGCTCAGTCATAGCA AAGAAGAATGAAAGATGGCATTCAGATGGAATCGTGTATCCCAAACCCACGTGGCTTGGAGAAGAGTTGC TGGCCAAGTTGGCCAAGTGGTCTGTAGAGAACAAGAAGAGTGACTTTAAAAGCACCCTTTCCCTCATCTC 10 CATTATGAAGTATAGCAAGGCTTACCAGGAACTTAAAGAGAAGTATAAGGAAATGGTTAAGGTGTGGCCT GAAGTCACTGATCCTGAGAAGTTCGTGTATGAAGATGTGGCTATCGCAGCATACCTGCTGATTCTATGGG AAGAAGAAAGGGCTGAGAGGAGACTAACTGCCAGGCAGTCCTTTGTGGACCTGGGATGTGGAAATGGCCT CCTGGTCCACATCCTGAGCAGTGAGGGGCATCCAGGCAGAGGGATTGATGTCCGAAGAAGAAAAATCTGG GACATGTATGGACCACAAACTCAGTTAGAGGAAGATGCAATCACACCCAATGATAAGACCCTTTTCCCTG 15 ATGTTGATTGGTTAATCGGTAACCATTCTGATGAACTCACACCATGGATACCTGTCATTGCAGCCAGGTC TTCCTACAATTGCCGCTTCTTTGTCCTCCCCTGCTGCTTCTTTGACTTCATTGGAAGATACTCCCGGAGG CAGAGTAAGAAGACTCAGTACCGGGAATACCTTGACTTCATTAAAGAAGTGGGCTTCACCTGTGGGTTTC ACGTGGACGAAGACTGCCTCAGGATTCCTTCAACCAAAAGAGTCTGTCTCGTTGGAAAATCCAGAACATA CCCTTCCTCCAGAGAAGCTTCCGTGGATGAAAAGAGGACTCAGTACATTAAGAGCAGGCGGGGCTGCCCT 20 GTAAGCCCACCTGGCTGGGAGCTTTCCCCTTCTCCACGCTGGGTTGCTGCTGGCAGTGCTGGTCACTGTG ACGGTCAGCAAGCTCTGGACGCCAGGGTCGGGTGTGTAACCAGGGCCTGGGCCGCTGAGCATGGAGCAGG GCCCCAGGCTGAAGGACCCTGGCTACCTGGATTTCATCCCAGAGAAAAGGCTGAGCGTGTGAGGAACTGT GCCGCCCTGCCACGAGATTTTATTGACCAAGTGGTTTTGCAAGTAGCGAATTTACTGTTAGGTGGAAAGC AATTAAACACAAGAAGTTCTCGAAATGGGAGTTTGAAGACCTGGAATGGGGGAGAGAGCCTATCTCTGGC 25 AGAAGTAGCCAACGAGCTGGACACGGAGACCCTGCGGAGGCTGAAGCGGGAGTGTGGGGGCCTGCAGACG CTGCTCCGGAACAGCCACCAGGTGTTCCAAGTTGTGAATGGGAGAGTTCACATCCGCGACTGGCGAGAGG AGACACTGTGGAAGACAAAGCAACCGGAAGCGAAACAGAGACTGCTCTCTGAAGCCTGCAAAACCCGCCT CTGCTGGTTCTTCATGCATCACCCTGATGGCTGCGCTCTGTCCACGGACTGCTGCCCGTTTGCCCATGGG CCTGCGGAGCTGCGGCCACCCCGGACCACCCCGAGGAAGAAGATTTCATGAGCTGCATCCTTGCCAGCCG 30 AGGCCTGGTTGGGGAGGCCAAACCAAGGAGAGCTTCCCCAGCAGTCGTCAGTGCTGTGGTCTCTGCTCTG GCTGTGTTTCAGCCCACCTCCTCCCAGCTTTCTCCACATCCTCACAGTGATGAACCGTATTTCATAAACA TCACACGCCAGAGAAGCCACAGTTACTCGGAAGCCCCCAGCTGACTGCCTGGCTTGTTTCAGATGCAGCC GCTTGAAACGTGCGCAGCATCTTCATATCATAAAGATTGTGCACGGATCCTTACAATGTCTCCTGGGGGA GAGCGGCTGAGGCTGCCTTGCACAGGCCCTTCCCAGGGCGCTGTCCGACGCCTGCCCCACCATGTCCACA 35 TCTGTGAAGAGGATGGGGCTCCTCGAGAAGTAAGACCGTATCTGCCAGCGTTTCTCACCACACTGGAGAG CAGCTGCTCTGGAGCAGGGATCCACCAGATTGGTATTTTTAAAAAAGGTGTCAGGCTTGCTATGTTGAGG TTGTTTTTAGAGTTACAGAGAATAAAAACACTCATAATTTCCTGA >NM_152544.3 Homo sapiens tRNA methyltransferase 44 homolog (TRMT44), 40 transcript variant 2, mRNA GTCATCTCGGCGCGCCGCTGCCAGGGCTGTACACCTGCTGGCTGCCATGGCTGAGGTGGGCCGTACCGGG 76 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 ATCAGCTACCCAGGCGCGCTTCTCCCACAGGGCTTCTGGGCTGCGGTCGAAGTGTGGCTGGAGAGGCCGC AGGTGGCAAACAAACGGCTTTGCGGCGCCCGCCTGGAGGCCCGCTGGAGCGCCGCCCTGCCCTGCGCGGA GGCCCGCGGCCCCGGGACTAGCGCAGGCTCGGAGCAGAAGGAGCGGGGTCCGGGACCCGGCCAGGGTTCC CCCGGAGGGGGCCCGGGTCCCAGGTCGCTATCAGGACCCGAGCAGGGCACGGCATGTTGCGAACTTGAGG 5 AGGCCCAGGGCCAGTGCCAGCAAGAGGAGGCACAGAGGGAAGCCGCCTCAGTGCCCCTGAGGGACTCCGG GCACCCCGGCCATGCTGAAGGAAGGGAGGGCGACTTCCCCGCCGCAGATCTGGATTCGCTTTGGGAGGAT TTCTCCCAAAGTCTCGCCCGTGGCAATTCGGAGTTGCTGGCCTTCCTCACCAGCTCCGGGGCGGGATCGC AGCCAGAGGCGCAGCGTGAGCTCGACGTGGTTCTCAGAACCGTCATCCCGAAAACTAGCCCACATTGCCC CCTTACAACTCCCAGGAGGGAAATAGTCGTGCAAGATGTCCTCAATGGAACCATAACGTTTTTGCCTTTG 10 GAAGAAGATGATGAGGGGAACCTAAAGGTTAAGATGAGCAATGTGTATCAAATTCAGCTCAGTCATAGCA AAGAAGAATGGTTCATATCTGTTTTAATTTTCTGTCCAGAAAGATGGCATTCAGATGGAATCGTGTATCC CAAACCCACGTGGCTTGGAGAAGAGTTGCTGGCCAAGTTGGCCAAGTGGTCTGTAGAGAACAAGAAGAGT GACTTTAAAAGCACCCTTTCCCTCATCTCCATTATGAAGTATAGCAAGGCTTACCAGGAACTTAAAGAGA AGTATAAGGAAATGGTTAAGGTGTGGCCTGAAGTCACTGATCCTGAGAAGTTCGTGTATGAAGATGTGGC 15 TATCGCAGCATACCTGCTGATTCTATGGGAAGAAGAAAGGGCTGAGAGGAGACTAACTGCCAGGCAGTCC TTTGTGGACCTGGGATGTGGAAATGGCCTCCTGGTCCACATCCTGAGCAGTGAGGGGCATCCAGGCAGAG GGATTGATGTCCGAAGAAGAAAAATCTGGGACATGTATGGACCACAAACTCAGTTAGAGGAAGATGCAAT CACACCCAATGATAAGACCCTTTTCCCTGATGTTGATTGGTTAATCGGTAACCATTCTGATGAACTCACA CCATGGATACCTGTCATTGCAGCCAGGTCTTCCTACAATTGCCGCTTCTTTGTCCTCCCCTGCTGCTTCT 20 TTGACTTCATTGGAAGATACTCCCGGAGGCAGAGTAAGAAGACTCAGTACCGGGAATACCTTGACTTCAT TAAAGAAGTGGGCTTCACCTGTGGGTTTCACGTGGACGAAGACTGCCTCAGGATTCCTTCAACCAAAAGA GTCTGTCTCGTTGGAAAATCCAGAACATACCCTTCCTCCAGAGAAGCTTCCGTGGATGAAAAGAGGACTC AGTACATTAAGAGCAGGCGGGGCTGCCCTGTAAGCCCACCTGGCTGGGAGCTTTCCCCTTCTCCACGCTG GGTTGCTGCTGGCAGTGCTGGTCACTGTGACGGTCAGCAAGCTCTGGACGCCAGGGTCGGGTGTGTAACC 25 AGGGCCTGGGCCGCTGAGCATGGAGCAGGGCCCCAGGCTGAAGGACCCTGGCTACCTGGATTTCATCCCA GAGAAAAGGCTGAGCGTGTGAGGAACTGTGCCGCCCTGCCACGAGATTTTATTGACCAAGTGGTTTTGCA AGTAGCGAATTTACTGTTAGGTGGAAAGCAATTAAACACAAGAAGTTCTCGAAATGGGAGTTTGAAGACC TGGAATGGGGGAGAGAGCCTATCTCTGGCAGAAGTAGCCAACGAGCTGGACACGGAGACCCTGCGGAGGC TGAAGCGGGAGTGTGGGGGCCTGCAGACGCTGCTCCGGAACAGCCACCAGGTGTTCCAAGTTGTGAATGG 30 GAGAGTTCACATCCGCGACTGGCGAGAGGAGACACTGTGGAAGACAAAGCAACCGGAAGCGAAACAGAGA CTGCTCTCTGAAGCCTGCAAAACCCGCCTCTGCTGGTTCTTCATGCATCACCCTGATGGCTGCGCTCTGT CCACGGACTGCTGCCCGTTTGCCCATGGGCCTGCGGAGCTGCGGCCACCCCGGACCACCCCGAGGAAGAA GATTTCATGAGCTGCATCCTTGCCAGCCGAGGCCTGGTTGGGGAGGCCAAACCAAGGAGAGCTTCCCCAG CAGTCGTCAGTGCTGTGGTCTCTGCTCTGGCTGTGTTTCAGCCCACCTCCTCCCAGCTTTCTCCACATCC 35 TCACAGTGATGAACCGTATTTCATAAACATCACACGCCAGAGAAGCCACAGTTACTCGGAAGCCCCCAGC TGACTGCCTGGCTTGTTTCAGATGCAGCCGCTTGAAACGTGCGCAGCATCTTCATATCATAAAGATTGTG CACGGATCCTTACAATGTCTCCTGGGGGAGAGCGGCTGAGGCTGCCTTGCACAGGCCCTTCCCAGGGCGC TGTCCGACGCCTGCCCCACCATGTCCACATCTGTGAAGAGGATGGGGCTCCTCGAGAAGTAAGACCGTAT CTGCCAGCGTTTCTCACCACACTGGAGAGCAGCTGCTCTGGAGCAGGGATCCACCAGATTGGTATTTTTA 40 AAAAAGGTGTCAGGCTTGCTATGTTGAGGTTGTTTTTAGAGTTACAGAGAATAAAAACACTCATAATTTC CTGA 77 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 By “vector” is meant a nucleic acid molecule, for example, a plasmid, cosmid, virus, or bacteriophage that is capable of replication in a host cell. In one embodiment, a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, 5 bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell. Typically, expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers. Unless specifically stated or obvious from context, as used herein, the term “about” is 10 understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. The recitation of a listing of chemical groups in any definition of a variable herein 15 includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. 20 BRIEF DESCRIPTION OF THE DRAWINGS FIGs.1A-1H provide a drawing, graphs, plots, a picture, and a heat map showing that 2’- O-methylation (Nm) at internal sites on mRNA and the expression of Nm writer protein FBL is 25 required for the greater stability of 2′-O-methylated mRNAs compared to the rest of mRNAs in HEK293T cells. FIG.1A shows Nm (top panel) and related questions on RNA stability (bottom). FIG.1B is a gene set enrichment analysis (GSEA) showing a relationship between 2′-O- Methylation and mRNA half-life. FIG.1C is a cumulative graph and boxplot to show the half- life of mRNAs with or without detectable Nm. Boxplot to show the half-life of mRNAs with or 30 without Nm. The top line in the graph is Nm(-) and the bottom line is Nm(+). FIG.1D is a Western blot to show the knockdown efficiency of FBL and NOP56 in C4-2 cells. FIGs.1E-1F show the cumulative fraction of mRNAs plotted against the change of mRNA half-life in response to knockdown of FBL (FIG.1E) and NOP56 (FIG.1F). In each of FIGs.1E-1F, the top line is Nm(+) and the bottom line is Nm(-). FIG.1G is a boxplot to show the half-life of 2′- 35 O-methylated mRNAs under individual conditions. FIG.1H is a heatmap to show half-life of top 78 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 2′-O-methylated mRNAs ranked by degree of half-life decrease upon FBL knockdown. P values were determined by two-tailed unpaired Wilcoxon’s test (boxplot in FIGs.1C, 1G) and K-S test (cumulative plot in FIGs.1C, 1E, 1F). All presented data were from HEK293T cells. RNA half- life determined based on public (FIGs.1B-1C) and the new (FIGs.1E-1H) RNA-seq data 5 generated following ActD inhibition of transcription. FIGs.2A-2J provide graphs, plots, a schematic, and drawings showing that the binding of FBL is required for the greater stability of FBL-binding mRNAs when compared to the rest of the mRNAs in HEK293T cells. FIG.2A is a bar plot showing the overlap of 2′-O-methylated mRNAs and FBL-binding mRNAs. The top portion of each bar shows percentage of no binding, 10 and the bottom portion shows percentage binding. FIG.2B shows the cumulative fraction of mRNAs plotted against half-life of mRNAs with or without detectable binding of FBL. The top line is FBL-binding(-) and the bottom line is FBL-binding(+). The inset box plot shows FBL- binding(-) on the right, and FBL-binding(+) on the left. FIG.2C shows the cumulative fraction of mRNAs plotted against half-life of mRNAs with high or low binding intensity of FBL. The 15 top line is FBL-binding low and the bottom line is FBL-binding high. The inset box plot shows FBL-binding low on the right, and FBL-binding high on the left. FIGs.2D-2E show the cumulative fraction of mRNAs plotted against the change of mRNA half-life in response to knockdown of FBL (FIG.2D) and NOP56 (FIG.2E). The top line in each graph is FBL- binding(+) and the bottom line is FBL-binding(-). FIG.2F is a boxplot to show the half-life of 20 FBL-binding mRNAs under three different conditions. FIG.2G is an RTL-P assay which detected Nm levels on four mRNAs (ACTG1, SNHG5, CALR, PHGDH) under control, siFBL, and siNOP56 conditions. The cartoon in the top left subpanel indicates the locations of primers used for RTL-P assay; m: Nm site; Um: upstream of Nm site; Dm: downstream of Nm site; F: forward primer; R: reverse primer; RT: Reverse transcription. FIG.2H is a densitometric 25 analysis of data from FIG.2G as shown as the signal intensity ratio of PCR products at low dNTP (1 μM) over high dNTP (1 mM) conditions. The ratio in control cells was set close to 1. Data represent Mean ± SD from n=3 biologically independent experiments. **, P < 0.01, ***, P < 0.001. P values were calculated based on a two-tailed Student’s t-test. FIG.2I is the relative expression level of mRNA plotted against time after inhibition of transcription in each panel. 30 The lines in each graph are ordered from top to bottom, as follows: siCTRL, siNOP56, siFBL. Expression was determined by RNA-Seq and normalized so that the value before inhibition of transcription was the same in all samples. (J) Bar plot to show steady-state mRNA expression level determined by RNA-Seq. **, P < 0.01; ***, P < 0.001 is calculated by edgeR. P values were determined by a two-tailed K-S test (FIGs.2B, 2C, 2D, 2E). All presented data were from 79 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 HEK293T cells. RNA half-life determined based on public (FIGs.2A-2C) and the new (FIGs. 2E-2F) RNA-seq data generated following ActD inhibition of transcription. FIGs.3A-3J are plots, graphs, and a pie chart showing that elevated FBL expression correlated with expression upregulation of FBL-binding and 2’-O-methylated mRNAs in 5 prostate cancer (PCa)( PCa) cells. FIG.3A is a boxplot showing RNA expression level of FBL, NOP56, and NOP58 in prostate normal and cancer samples from the TCGA project. FIG.3B shows that the genome browser tracks showing RNA-Seq read density at individual gene loci of C4-2 and PrEC cells. FIG.3C is a pie chart showing the distribution of FBL-binding sites in individual categories of RNAs in C4-2 cells. FIG.3D is a GSEA result which shows the 10 relationship between the binding of FBL on mRNA and the expression change of mRNA in response to FBL knockdown. FIG.3E is a boxplot to show expression change of mRNAs in response to FBL knockdown. FIG.3F shows the cumulative fraction of mRNAs plotted against the change of mRNA half-lives in response to FBL knockdown in C4-2 cells. The top line is FBL-binding(+) and the bottom line is FBL-binding(-). FIG.3G is a boxplot to show mRNA 15 half-life of FBL-binding mRNAs under two conditions in C4-2 cells. FIG.3H shows the cumulative and boxplot of half-life change of mRNAs between C4-2 and PrEC cells. The top line is C4-2 FBL-binding(-) and the bottom line is C4-2 FBL-binding(+). FIG.3I is a GSEA to show the relationship between the binding of FBL on mRNA in C4-2 cells and the expression change of mRNA between C4-2 and PrEC cells. FIG.3J is a boxplot showing expression change 20 of mRNAs between C4-2 and PrEC cells. FBL-binding mRNAs in all figures were defined in the C4-2 control cells. P-values were determined by two-tailed unpaired Wilcoxon’s tests (FIGs.3A, 3E, 3G, boxplot in 3H, 3J) and K-S test (FIGs.3F, cumulative curve in 3H). FIGs.4A-4K are a schemtica, graphs, plots, and a pie chart showing a machine learning model for detection of Nm based on Nanopore direct RNA-seq. FIG.4A is a cartoon showing 25 the flowchart of a machine learning model to detect Nm based on Nanopore direct RNA-seq. FIG.4B are receiver operating characteristic (ROC) curves which demonstrate the performance of the machine learning model for detecting known Nm sites in the human thyroid carcinoma cell line of KTC-1. The two panels were plotted for two different 5-mers. FIGs.4C-4D are bar plots to show the Nm modification ratio defined by the machine learning model at individual 30 sites on the 28S (FIG.4C) and 18S (FIG.4D) rRNAs of C4-2 cells. FIGs.4E-4F are boxplots to show Nm modification ratio defined by the machine learning model at individual sites on 28S rRNA (FIG.4E) and 18S rRNA (FIG.4F) in C4-2 cells under siCTRL and siFBL conditions. FIG.4G shows rRNA Nm modification ratio at known snoRNA-targeted Nm sites on 25S rRNA in yeast under wild type, snR60 knockout, snR61 knockout, and snR62 knockout conditions. 80 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 The bars in each set of four are ordered, from top to bottom, as follows: WT; snR60_KO; snR61_KO; snR62_KO. FIGs.4H-4I is a boxplot to show Nm modification ratio defined by the machine learning model at individual sites on 28S rRNA (FIG.4H) and 18S rRNA (FIG.4I) in Drosophila cells under siCTRL and siFBL conditions. FIG.4J is a probability density plot of 5 Nm modification ratio for mRNAs and rRNAs in C4-2 cells. The left peak is mRNA and the right peak is rRNA. FIG.4K is a pie chart that shows the distribution of Nm sites in different sequence regions in C4-2 cells. P values were determined by two-tailed unpaired Wilcoxon’s tests (FIGs.4E, 4F, 4H, 4I). FIGs.5A-5M are graphs, plots, and a diagram showing that Nm detected on mRNA by 10 machine learning model based on Nanopore direct RNA-seq is downregulated by FBL knockdown in C4-2 cells. FIG.5A is a boxplot to show the Nm ratio of Nm sites on mRNAs. FIG.5B is a scatter plot to show the Nm ratio of individual Nm sites on mRNAs in control and FBL knockdown cells. FIG.5C is an average density plot of Nm sites on mRNAs. The top line is siCTRL and the bottom line is siFBL. FIG.5D is a Venn diagram to show overlap between15 FBL-binding and 2′-O-methylated mRNAs. FIG.5E is a bar plot that shows the percentage of 2’- O-methylated mRNAs for individual gene groups ranked by mRNA expression level from the lowest on the left side to the highest on the right side. FIG.5F is a GSEA that shows the relationship between mRNA Nm modification and expression change upon knockdown of FBL. FIG.5G is a boxplot showing expression change of individual mRNA groups upon FBL 20 knockdown. FIG.5H is a boxplot showing mRNA half-life of individual mRNA groups. FIG.5I shows the cumulative fraction of mRNAs plotted against degree of mRNA half-life change upon FBL knockdown. The lines are ordered, from top to bottom, as follows: Nm Hypo; Nm(+); Nm(-). FIG.5J is a boxplot showing mRNA half-life change of individual mRNA groups in response to knockdown of FBL. FIG.5K is a boxplot showing the half-life of 2′-O-methylated25 and unmethylated noncoding RNAs. FIG.5L is a boxplot showing expression change of 2′-O- methylated noncoding RNAs in C4-2 cells. FIG.5M is a boxplot showing mRNA half-life of noncoding RNAs 2′-O-methylated in C4-2 cells. P values were determined by two-tailed unpaired Wilcoxon’s tests (FIGs.5A, 5G, 5H, 5J-M) and K-S test (FIG.5I). All presented data were from C4-2 cells. 30 FIGs.6A-6P are heatmaps, graphs, plots, and pictures showing that FBL expression in PCa cell C4-2 is required for the Nm modification and expression of mRNAs in cancer pathways. FIG.6A is a heatmap to show Nm ratio of 10 example Nm sites (on 8 mRNAs) that show down-regulation of Nm in Nanopore direct RNA-seq data upon FBL knockdown in C4-2 cells. FIG.6B is an RTL-P assay to detect the Nm level at 7 Nm sites of mRNAs shown in (FIG. 81 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 6B). Total RNAs from control, FBL-deficient, and NOP56-deficient C4-2 cells were extracted and subjected to reverse transcription (RT) with RT primer at low (1 μM) or high (1 mM) concentration of dNTP, respectively. The obtained cDNA was then amplified with primer pairs corresponding to upstream (Um) or downstream (Dm) regions of the methylation site. FIG.6C is 5 a densitometric analysis of data from FIG.6B shown as signal intensity ratio of PCR products at low dNTP (1 μM) over high dNTP (1 mM) level. Methylation levels in control cells were set close to 1. Data represent Mean ± SD from n=3 biologically independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 is based on two-tailed Student’s t-test. FIG.6D is a heat map to show the mRNA expression level of the 8 example genes. FIG.6E is a boxplot showing 10 expression change of C4-2 cell 2′--O-methylated mRNAs in PCa and normal patients from the TCGA project dataset. FIG.6F is a Western blot showing the protein levels of PSMD13 in each group as indicated. FIGs.6G-6H are RTL-P assays to detect the Nm level at two Nm sites of PSMD13 in each group as indicated. FIG.6I is a boxplot showing the expression of PSMD13 in the TCGA prostate cancer patients grouped based on nodal metastasis status. FIG.6J shows 15 disease-free survival of patients with different PSMD13 expression levels in PCa samples from the TCGA dataset. The top line is Low and the bottom line is High. FIG.6K is a Western blot to show the knockdown efficiency of PSMD13 in C4-2 cells. FIG.6L is a CellTiter assay to measure the effect of PSMD13 knockdown on cell proliferation. The lines are ordered, from top to bottom, as follows: siCTRL; siPSMD13-1; siPSMD13-2. FIGs.6M-6N is a transwell assay to 20 measure the change of cell invasion capability upon PSMD13 knockdown (n=8). FIGs.6O-6P is a Wound Healing assay to determine the change of cell migratory capability upon PSMD13 suppression (n=6). All presented data were from C4-2 cells except the patient samples from the TCGA database. *P < 0.05, **P < 0.01, ***P < 0.001 is based on the student’s t-test unless otherwise stated. 25 FIGs.7A-7N are graphs, plots, a diagram, a picture, and a drawing showing that mRNA stabilization by Nm is associated with 3′ UTR shortening. FIG.7A is a cumulative fraction plot of 3′ UTR lengths of individual mRNAs in three groups. The lines are ordered, from top to bottom, as follows: Nm Hypo; Nm(+); Nm(-). FIG.7B is a box plot to show the number of miRNA target sites in 3′ UTR regions of individual mRNAs in three groups. FIG.7C is a box 30 plot to show ARE scores for 3′ UTR regions of individual mRNAs in three groups. FIG.7D is a scatter plot to show PDUI values of individual mRNAs in control and FBL knockdown conditions. The internal bar plot shows numbers of 3′ UTR lengthening and shortening events induced by FBL knockdown. FIG.7E is a Venn diagram that shows the overlap between genes showing lengthened 3′UTR upon siFBL and 2′-O-methylated genes under control condition. 82 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 FIG.7F is a bar plot to show the number of overlaps observed or expected by chance between genes that show lengthened or shortened 3′ UTR upon siFBL and genes that show 2′-O- methylated mRNAs under control condition. The left bar in each set of two is Overlap, and the right bar is Expected. FIG.7G is a box plot to show mRNA Nm ratios of individual genes that 5 showed lengthened and shortened 3′UTR upon knockdown of FBL. The ratio of unmethylated sites in siFBL was considered to be 0. FIG.7H is a cumulative fraction plot shows mRNA half- life changes of individual gene groups that show lengthened and shortened 3’ UTRs upon FBL knockdown. The top line is lengthen and the bottom line is shorten. FIG.7I shows genome browser overlay line tracks of RNA-seq read density of four genes that show 3′ UTR 10 lengthening, hypo-methylation, and decreased mRNA stability upon knockdown of FBL. Blue and red curve lines are plotted for control and FBL-knockdown cells, respectively. Grey and black vertical lines show Nm sites hypomethylated and do not change significantly upon FBL knockdown, respectively. FIG.7J is a bar plot that shows the percentage of Nm sites overlapping with the binding regions of 10 different RNA binding proteins (RBPs) in HEK293T 15 cells. The top bar in each set of two is Overlap, and the bottom bar is Expected. FIG.7K is a scatter plot showing a correlation between the PDUI changes induced by FBL and CPSF depletion in HepG2 cells. FIG.7L shows RIP-qPCR to detect the change of the binding of CPSF7 to 2′-O-methylated mRNAs upon FBL knockdown. The bars in each set of three are ordered, from left to right, as follows: siCTRL; siFBL-1; siFBL-2. FIG.7M is a Western blot to 20 show the expression of individual proteins under individual conditions. FIG.7N is a cartoon to show the working model of Nm-dependent regulation of RNA stability. P values were determined by two-tailed unpaired K-S test (FIG.7A), Wilcoxon test (FIGs.7B, 7C, 7G), two- tailed binomial test (FIG.7D), one-tailed Fisher’s exact test, (FIG.7F) one-tailed unpaired K-S test (FIG.7H), Fisher exact test(FIG.7J), Pearson correlation test (FIG.7K), and t-test (FIG. 25 7L). All presented data are from C4-2 cells unless labeled explicitly as from HepG2 cells. Hypo, mRNAs showing hypomethylated Nm sites upon FBL knockdown in C4-2 cells. Nm (+), mRNAs showing Nm sites in control C4-2 cells. Nm (-), mRNAs showing no detectable Nm sites in control C4-2 cells. FIGs.8A-8I provide a flow chart, graphs, a diagram, plots, and a heatmap showing an 30 analysis of half-life, Nm, and binding of FBL in mRNAs of HEK293T cells. FIG.8A is a flow chart of RNA-seq experiment to profile RNA half-life. FIG.8B is a bar plot that shows the expression of FBL and NOP56 determined by RT-qPCR in the control and knockdown cells. FIG.8C shows that the genome browser tracks showing RNA-seq read density at the FBL and NOP56 loci. FIG.8D is a Venn diagram showing the overlap between FBL-binding and 2′-O- 83 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 methylated mRNAs. FIG.8E is a cumulative plot and boxplot showing half-life of 2′-O- methylated and unmethylated mRNAs. The top line is Nm(+) and the bottom line is Nm(-). FIG.8F is a boxplot showing half-life of mRNA with or without detectable FBL binding. FIG. 8G is a boxplot showing half-life of mRNAs with high or low binding intensity of FBL. FIG.8H 5 is a heatmap to show half-life of FBL-binding mRNAs; 50 mRNAs ranked on top based on the degree of half-life decrease upon FBL knockdown were shown. FIG.8I shows Genome browser tracks showing RNA-seq read density at the loci of 2′-O-methylated mRNAs. P values were determined by two-tailed Student’s t-test (FIG.8B), Fisher’s exact test (FIG.8D), two-tailed unpaired Wilcoxon’s tests (boxplot in FIGs.8F, 8G, 8H), and K-S test (cumulative plot in FIG. 10 8F). All presented data were from HEK293T cells. RNA half-life determined based on the new (FIG.8E-8H) RNA-seq data generated following ActD inhibition of transcription. FIGs.9A-9H provide plots, graphs, a drawing, a diagram, and a pie chart showing that protein members of the Box C/D snoRNP tend to show up-regulated RNA expression in cancer samples relative to control samples and bind mRNAs in C4-2 cells. Boxplot showing RNA 15 expression of the snoRNP proteins FBL (FIG.9A), NOP56 (FIG.9B), and NOP58 (FIG.9C) in cancer and normal patient samples of individual cancer types in the TCGA dataset. In each figure, for each set of two boxes, the left box is Normal, and the right box is Tumor. FIG.9D is a flowchart of RIP-seq experiment to detect FBL-binding RNAs. FIG.9E is a Venn diagram showing the overlap of FBL-binding mRNAs between C4-2 and HEK293T cell lines. FIG.9F is 20 a pie chart that shows the percent of FBL-binding small nucleolar RNAs (snoRNAs) that have C/D box or not in C4-2 cells. The pie chart indicates that N=102 for C/D box and N=4 for Others. FIG.9G shows that the IGV genome browser shows examples of C/D box snoRNAs bound by FBL in C4-2 cells. FIG.9H shows that the IGV genome browser shows examples of mRNAs bound by FBL in C4-2 cells. P value was calculated by Fisher’s exact test (FIG.9E).25 FIG.10A-10G provide a flowchart, plots, graphs, and piecharts showing a machine- learning strategy to detect Nm modification based on Nanopore direct RNA-seq. FIG.10A shows the computational pipeline for detecting RNA Nm sites from Nanopore directive RNA- seq. FIGs.10B, 10C are bar plots showing the accuracy (FIG.10B) and AUROC (FIG.10C) of the machine learning model when applied to each 1005-mers in KTC-1 cells. FIG.10D is a bar30 plot showing RNA expression level of FBL determined by Nanopore RNA-seq in individual C4- 2 samples. FIG.10E is a double pie chart to show the overlap of the detected rRNA Nm sites in C4-2 cells and the known other types of modification sites in rRNAs from literature (Taoka, M., Nobe, Y., Yamaki, Y., Sato, K., Ishikawa, H., Izumikawa, K., Yamauchi, Y., Hirota, K., Nakayama, H., Takahashi, N., and Isobe, T. (2018). Landscape of the complete RNA chemical 84 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 modifications in the human 80S ribosome. Nucleic Acids Res 46, 9289-9298. 10.1093/nar/gky811). FIG.10F is a nested pie chart to show percentages of A, U, G, and C bases among detected Nm sites in C4-2 cells (outer), all bases in mRNAs (middle), and bases in the 5-mers covered by the machine-learning model (inner). The sections of the chart are as 5 follows, from the top moving clockwise: U; C; A; G. FIG.10G is an average density plot of Gm, Cm, Um, and Am sites across the transcript body of mRNAs in siCTRL and siFBL C4-2 cells. The top line in each graph is siCTRL and the bottom line is siFBL. FIG.11A-11R provide graphs, plots, a diagram, and a chart showing that FBL promotes widespread Nm in mRNAs with increased RNA stability in the cells. FIG.11A is a bar plot to 10 show enrichment of KEGG pathways in 2′-O-methylated mRNAs of C4-2 cells. FIG.11B is a GSEA to demonstrate the relationship between Nm in C4-2 cells and the expression change of mRNAs between C4-2 and PrEC cells. FIG.11C is a cumulative plot and boxplot showing expression change of 2′-O-methylated mRNAs between C4-2 and PrEC cells. The top line is C4-2 Nm(+) and the bottom line is C4-2 Nm(-). 2′-O-methylated mRNAs were defined in the 15 C4-2 cells. FIG.11D is a boxplot showing expression of mRNAs 2′-O-methylated in C4-2 cells. FIG.11E is a flow chart of the SLAM-seq experiment to profile RNA half-life. FIGs.11F-11G are boxplots showing the T to C transition rate in samples at individual time points of SLAM-seq in siCTRL (FIG.11F) and siFBL (FIG.11G) C4-2 cells. FIG.11H is a boxplot showing mRNA half-life of individual mRNA groups in C4-2 cells. FIG.11I is a cumulative plot to show mRNA 20 half-life change in response to knockdown of FBL in C4-2 cells. FIG.11J is a boxplot showing mRNA half-life change of individual mRNA groups in response to FBL knockdown in C4-2 cells. FIG.11K is a boxplot showing half-life of mRNAs with FBL binding or no FBL binding in C4-2 cells. FIG.11L is a cumulative plot showing mRNA half-life change upon FBL knockdown in C4-2 cells. The top line is FBL-binding (+) and the bottom line is FBL-binding (- 25 ). FIG.11M is a boxplot showing half-life of FBL-binding mRNA in the control and FBL knockdown C4-2 cells. FIG.11N is a Venn diagram showing the overlap between FBL-binding and 2′-O-methylated transcripts in HEK293T cells. FIG.11O is a cumulative plot and boxplot plot showing half-life of 2′-O-methylated and unmethylated mRNAs in HEK293T cells. The top line is Nm(-) and the bottom line is Nm(+). FIGs.11P-11Q are cumulative plots showing half- 30 life change of mRNAs upon knockdown of FBL (FIG.11P) and NOP56 (FIG.11Q) in HEK293T cells. FIG. The top line in each graph is Nm(+) and the bottom line is Nm(-). 11R is a boxplot showing half-life of mRNAs 2′-O-methylated in HEK293T cells. P values were determined by two-tailed unpaired Wilcoxon’s tests (FIGs.11D, 11H, 11J, 11K, 11M, boxplot in 11O) and K-S test (FIGs.11B, 11I, 11L, cumulative plot in 11O, 11P-Q). The half-life of 85 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 mRNAs was determined by SLAM-Seq (FIG.11E-11M) and RNA-seq following transcription inhibition by ActD (FIG.11O-11R). Hypo: mRNA hypomethylated upon FBL knockdown; Nm (+): 2′-O-methylated mRNA in control C4-2 cells (FIG.11A-11M) or HEK293T cells (FIG. 11O-11R); Nm (-): unmethylated mRNA in control C4-2 cells (FIG.11A-11M) or HEK293T 5 cells (FIG.11O-11R). FBL-binding mRNAs were defined in control C4-2 cells (FIG.11A-11M) or HEK293T cells (FIG.11O-11R). FIGs.12A-12W provide plots, graphs, a heatmap, and a diagram showing that stabilization of mRNA by Nm is not a confounding effect of RNA expression level, translation efficiency, or other known types of RNA modification. FIG.12A is a box plot showing 10 translation efficiency change of 2′-O-methylated (Nm (+)) and unmethylated (Nm (-)) mRNAs upon FBL knockdown in C4-2 cells. FIGs.12B-12F are boxplots showing mRNA translation efficiency (FIG.12B), mRNA half-life determined by transcription inhibition method (FIG. 12C), mRNA half-life change determined by transcription inhibition method (FIG.12D), mRNA half-life determined by SLAM-seq method (FIG.12E), and mRNA half-life change determined 15 by SLAM-seq method (FIG.12F) in C4-2 cells. FIGs.12G, 12J show numbers of Nm sites at different codon positions and the associated mRNAs in C4-2 cells (FIG.12G) and HEK293T cells (FIG.12J). The left bar in each set of two in each graph represents sites and the right bar represents gene. FIGs.12H-I, K are boxplots showing mRNA half-life determined by transcription inhibition method in C4-2 cell (FIG.12H) and HEK293 cell (FIG.12K) and 20 SLAM-seq method in C4-2 cells (FIG.12I). FIGs.12L-12N are boxplots showing expression level (FIG.12L), half-life determined by transcription inhibition method (FIG.12M), and half- life determined by SLAM-seq method (FIG.12N) for 2′-O-Methylated and unmethylated mRNAs in C4-2 cells. FIGs.12O-12P are boxplots showing expression level (FIG.12O) and half-life (FIG.12P) of mRNAs bound or not bound by FBL in HEK293T cells. mRNA half-life 25 is determined by the transcription inhibition method. FIG.12Q is a heatmap showing mRNA relative half-life values in C4-2 cells. FIGs.12R-S are a Venn diagram (FIG.12R) and bar plot (FIG.12S) showing the overlap between mRNAs with decreased half-life upon FBL knockdown and 2′-O-methylated mRNAs in C4-2 cells. mRNA half-life is determined by the transcription inhibition method. FIGs.12T-12U are bar plots showing the overlap between Nm sites detected 30 by our method and the Nm-mut-seq method in HeLa (FIG.12T) and HepG2 (FIG.12U) cells. FIG.12V is a bar plot showing the overlap of Nm sites detected by our method and sites of several other modification types in HEK293T cells. The left bar in each set of two is Overlap, and the right bar is Expected. FIG.12W is a scatter plot showing expression correlation between FBL and PSMD13 in the GTEx database. 86 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 FIGs.13A-13W provide plots, graphs, and a diagram showing the sequence features of methylated and unmethylated transcripts in C4-2 and HEK293T cells. FIGs.13A-13H are box plot to show the 5′ UTR length (FIG.13A), number of miRNA-binding motifs in 5′ UTR (FIG. 13B), CDS length (FIG.13C), number of miRNA-binding motifs in CDS (FIG.13D), intron 5 length (FIG.13E), 5′ UTR GC content (FIG.13F), 3′ UTR GC content (FIG.13G), and CDS GC content (H) of individual gene groups. Hypo, mRNAs showing hypomethylated Nm sites upon FBL knockdown in C4-2 cells. Nm (+), mRNAs showing Nm sites in control C4-2 cells. Nm (-), mRNAs showing no detectable Nm sites in control C4-2 cells. FIG.13I is a Venn diagram to show overlap between genes that have shortened 3′ UTR upon siFBL and genes bearing 2′-O- 10 methylated mRNAs in the control. FIG.13J shows Genome browser tracks to show RNA-seq read density of individual genes in C4-2 cells. FIGs.13K-13U are box plots to show 3′ UTR length (FIG.13K), number of potential miRNA-binding motifs in 3′ UTR (FIG.13L), 3′ UTR ARE score (FIG.13M), 5′ UTR length (FIG.13N), number of miRNA-binding motifs in 5′ UTR (FIG.13O), CDS length (FIG.13P), number of miRNA-binding motifs in CDS (FIG.13Q), 15 intron length (FIG.13R), 5′ UTR GC content (FIG.13S), 3′ UTR GC content (FIG.13T), and CDS GC content (FIG.13U) of individual gene groups. Nm (+), mRNAs showing Nm sites in control HEK293T cells. Nm (-), mRNAs showing no detectable Nm sites in control HEK293T cells. FIGs.13V-13X show Length of the 3′ UTR (FIG.13V), mRNA half-life (FIG.13W) with Nm only happened in CDS and only occurred in 3′ UTR in C4-2 cells. P values were determined 20 by a two-tailed unpaired Wilcoxon test (FIGs.13A-13H, 13K-13W). FIGs.14A-14E provide graphs and plots showing RNA-binding proteins with a potential to mediate Nm function. FIG.14A shows distributions of the protein-binding Nm sites in different sequence regions of mRNA in HEK293T cells. For each bar, the sections of the bar are divided, from left to right, as follows: 3’-UTR; Exon; Non-Coding; Promoter; 5’-UTR; Intron. 25 FIG.14B shows enrichment of the binding regions for individual RNA-binding proteins around Nm sites in HEK293T cells. FBL and TNRC6C were shown as the positive and negative controls. FIG.14C is a boxplot showing mRNA half-life of CPSF7-binding transcripts under FBL knockdown and control conditions in HEK293T cells. The P value is determined by the two-tailed unpaired Wilcoxon test. FIGs.14D-14E show Genome browser tracks showing RNA- 30 seq read density at the 3′ UTR (FIG.14D) or gene body (FIG.14E) of each gene in HepG2 cells. 87 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 DETAILED DESCRIPTION OF THE INVENTION The disclosure features compositions comprising non-naturally occurring RNAs modified 5 at internal sites and methods for stabilizing RNA (e.g., mRNA). The disclosure is based, at least in part, on the discovery, described herein, that modification of RNA through 2’-O-methylation (Nm or 2'-O-Me) at internal sites within the RNA surprisingly increased the stability of the RNA. In some embodiments, the RNA is an mRNA modified to include Nm modifications at internal sites, at least in part through the 10 activity of fibrillarin (FBL). In some embodiments, the RNA is in vitro transcribed or chemically synthesized and then 2’-O-methyl modified. In some embodiments, the in vitro transcribed or chemically synthesized RNA comprises an increased number of Nm modifications relative to a naturally occurring RNA, or comprises Nm modifications at residues that are not modified in a naturally occurring RNA. In some embodiments, the in vitro transcribed or 15 chemically synthesized RNA is an mRNA, rRNA, tRNA, miRNA, snRNA, snoRNA, viral RNA, or RNA/DNA hybrid that comprises one or more Nm modifications. In one particular embodiment, the RNA is an mRNA comprising Nm modifications at 10%, 20%, 50%, 75% or 100% of ribonucleosides (e.g., internal sites). In the Examples described herein, the effect of Nm modification and the binding of FBL 20 on mRNA stability in both FBL- and NOP56-deficient cell lines was investigated, as compared to control cells. The regulation of mRNA Nm modification was then investigated based on a machine learning model, which utilized key features of known Nm sites in the rRNAs to detect new Nm sites based on direct RNA-seq data generated by the Oxford Nanopore Technologies™ (ONT) sequencer. In addition to accurately recapturing the well-known Nm sites in rRNA, the 25 model further identified about 30,000 internal Nm sites on mRNAs. Depletion of FBL was demonstrated to decrease the Nm levels as well as the stability and expression level of the mRNAs. The role of the binding of FBL and the Nm modification was investigated on post- transcriptional regulation of mRNA in prostate cancer (PCa) and normal cells. To understand how Nm affects mRNA stability, a comprehensive analysis was performed. A connection 30 between Nm and sequence-encoded features was observed, including 3′ UTR length, ARE (AU rich elements) score, miRNA binding sites, and GC content. For instance, it was found that Nm connected to alternative polyadenylation, which leads to 3′ UTR shortening and thus increased mRNA stability. By precisely measuring the stoichiometry of Nm at base resolution in mRNAs based on a combination of Nanopore direct RNA-seq and machine learning techniques, a novel 88 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 oncogenic role of FBL in Nm-mediated post-transcriptional regulation of mRNA stability was also revealed in cancer cells. These findings highlight the function of Nm in stabilizing mRNAs across the transcriptome. 5 RNA Modifications RNA modifications play pivotal roles in regulating RNA processing and translation (Roundtree et al. (2017), Cell 169, 1187-1200). Detecting RNA modifications at individual sites on each RNA molecule in the cell is a key step to understanding the function and regulation of these modifications. To date, however, the function of the Nm at mRNA internal sites in the 10 cells, e.g., the effect on mRNA stability and the underlying mechanism, is unclear. 2’-O-methylation (Nm) A particular type of RNA modification, 2'-O-methylation (2'-O-Me or Nm), stands out due to its widespread occurrence on all four types of nucleotides (A, U, G, C) and in most RNA 15 categories, e.g., mRNA, rRNA, tRNA, miRNA, snRNA, snoRNA, and viral RNA. Modified RNAs described herein include all of these categories of RNAs. Chemically, 2'-O-Me consists of a methyl group being added to the oxygen atom that is attached to the second carbon in the ribose sugar of a nucleotide (FIG.1A). The most prevalent site for 2'-O-Me in mRNA is the mRNA 5’ cap. This cap is a20 protective structure found at the 5’ end of eukaryotic mRNA and plays an important role in pre- processing and stabilizing mRNA. The 2'-O-Me modification on the first nucleotide in the mRNA cap, known as N12'-O-Me (Cap1), is specifically catalyzed by the enzyme cap methyltransferase 1 (CMTR1) that adds a methyl group onto the 2'-O-ribose of the first transcribed nucleotide. Meanwhile, CMTR2 is responsible for ribose 2'-O-Me at the second 25 transcribed nucleotide (Cap2). Cap 2'-O-Me plays a crucial role in masking mRNA from the innate immune surveillance mechanisms, preventing the recognition of mRNA as non-self or viral RNA. Hence, deregulation in CMTR1 expression naturally affects immune system-related pathways, inducing diseases such as asthma and Alzheimer’s (AD). 30 As the most abundant RNA modification on rRNAs, Nm modifications play an essential role during rRNA processing, ribosome biogenesis, and regulation of ribosome functions during translation. In sharp contrast to this well-known role on rRNA, the function of Nm at mRNA internal sites is currently unclear. It is well established that C/D-box small nucleolar RNAs guide RNA methyltransferase Fibrillarin (FBL), which forms a complex with other proteins such as the 89 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 NOP56, NOP58, and SNU13, to catalyze Nm on rRNA. In addition, FBL plays essential roles in many biological processes such as histone H2AQ104 methylation and eye and craniofacial development. Oncogenic roles of FBL in Nm of rRNAs were also reported, e.g., in prostate cancer (PCa) (Marcel et al., Cancer Cell 24, 318-330; Yi et al., Nat Cell Biol 23, 341-354) and 5 breast cancer (Su et el.,Oncogene 33, 1348-1358.10.1038/onc.2013.89). However, little is known about the oncogenic role of Nm at mRNA internal sites and the relevance of FBL in this modification. Most methods that detect RNA modification rely on either antibody, chemical treatment, or reverse transcription errors, which are powerful, but each also has some limitations, e.g., 10 introducing some biases and artifacts. As outstanding examples of such methods, the Nm-seq (Dai et al., Nat Methods 14, 695-698) and Nm-Mut-seq (Chen et al., Cell Res 33, 727-730) enabled transcriptome-wide detection of Nm sites and successfully revealed that Nm modifications were present at internal sites of mRNA. However, a quantitative way to directly measure the abundance of transcriptome-wide Nm modification with stoichiometric information 15 is still lacking. Nanopore direct RNA-seq represents a promising approach to concurrently detect multiple types of RNA modifications based on electrical signals of the nucleotides (Garalde, et al. (2018). Nat Methods 15, 201-206.10.1038/nmeth.4577; Wang et al., (2021). Nat Biotechnol 39, 1348-1365.10.1038/s41587-021-01108-x; Jain et al., (2022). Nat Methods 19, 1160-1164. 10.1038/s41592-022-01633-w). This cutting-edge method was successfully utilized to map RNA 20 modifications such as m6A (Liu et al., Nat Commun 10, 4079; , and A-to-I editing in different species . In terms of Nm, despite analysis of sequencing signals at known Nm sites in rRNA (Begik et al., Nat Biotechnol 39, 1278-1291; Stephenson et al., (2022). Cell Genom 2. 10.1016/j.xgen.2022.100097) and prediction of Nm sites as “yes” or “no” events without stoichiometry information in mRNA (Salem et al., (2022). Nm-Nano: Predicting 2′-O- 25 methylation (Nm) Sites in Nanopore RNA Sequencing Data. bioRxiv), there is yet no algorithm for de novo measurement of Nm stoichiometry using Nanopore direct RNA sequencing (Begik et al., Nat Biotechnol 39, 1278-1291). Modified Polynucleotides 30 Polynucleotides of the disclosure comprise modified ribonucleotides comprising a 2’-O- methyl modification that increases the stability of the polynucleotide. In some embodiments, the polynucleotide is an RNA molecule where at least about 10% of the internal sites (i.e., sites between the 5’ and 3’ termini comprise a 2’-O-methyl modification). In other embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the ribonucleotides of the RNA 90 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 are modified. In some embodiments, the RNA is a modified mRNA. In other embodiments, the RNA is chemically synthesized or in vitro transcribed. Modified mRNA refers to a nucleic acid molecule comprising at least one modified nucleoside. In some embodiments, a modified mRNA is generated by in vitro transcription or 5 chemical synthesis. In some embodiments, a modified mRNA is translatable in a mammalian (e.g., human) cell. In some embodiments, a modified mRNA is exhibits increased stability (e.g., resistance to degradation through enzymatic activity or other regulatory means and/or an extended half-life) in a cellrelative to a synthetic, non-modified RNA of the same sequence. In some embodiments, the modified RNA is generated by in vitro transcription of a DNA 10 template. Methods for generating RNAs are well known to those of skill in the art using standard molecular cloning techniques. The transcribed, modified mRNA polymer can be modified further post-transcription, e.g., by adding a cap or other functional group. To be suitable for in vitro transcription, the modified nucleoside(s) are recognized as substrates by at least one RNA polymerase enzyme expressed by the tissue or cell which is 15 transfected with the modified mRNA. Generally, RNA polymerase enzymes can tolerate a range of nucleoside base modifications, at least in part because the naturally occurring G, A, U, and C nucleoside bases differ from each other quite significantly. Thus, the structure of a modified nucleoside base for use in generating the modified mRNAs described herein can generally vary more than the sugar-phosphate moieties of the modified nucleoside. That said, ribose and 20 phosphate-modified nucleosides or nucleoside analogs are known in the art that permit transcription by RNA polymerases. In some embodiments of the aspects described herein, the RNA polymerase is a phage RNA polymerase. The modified nucleotides pseudouridine, m5U, s2U, m6A, and m5C are known to be compatible with transcription using phage RNA polymerases. Polymerases that accept modified nucleosides are known to those of skill in the art. 25 It is also contemplated that modified polymerases can be used to generate modified mRNAs, as described herein. Thus, for example, a polymerase that tolerates or accepts a particular modified nucleoside as a substrate can be used to generate a synthetic, modified mRNA including that modified nucleoside. In some embodiments, aa modified mRNA is translatable in vivo by the translation 30 machinery of a eukaryotic, preferably mammalian, and more preferably, human cell. Translation generally requires at least a ribosome binding site, a methionine start codon, and an open reading frame encoding a polypeptide. In some embodiments, the modified RNA RNA is an mRNA that comprises a 5' cap, a stop codon, a Kozak sequence, and/or a polyA tail. In some embodiments, the RNA is a non-coding mRNA. In still other embodiments, the RNA is a microRNA. 91 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Some nucleoside modifications alter the rate of translation. One can test a modified mRNA for its ability to undergo translation and translation efficiency using an in vivo translation assay (e.g., using a modified mRNA encoding a cre recombinase gene in an in vivo mouse cre model assay, or modified mRNA encoding luciferase and detecting expression in vivo using a 5 bioluminescence assay of the translated protein) and detecting the amount of the polypeptide produced using SDS-PAGE, Western blot, or immunochemistry, bioluminescence assays, etc. The translation of a modified mRNA comprising a candidate modification is compared to the translation of an RNA lacking the candidate modification. It is noted that fluoro-modified nucleosides are generally not translatable and can be used herein as a negative control for an in 10 vitro translation assay. Desirably, the modified mRNA has an increased half-life, and/or shows increased resistance to degradation through enzymatic or other regulatory means (e.g., in vivo, such as in a cell), as compared to an unmodified mRNA. 15 Methods for Increasing mRNA Stability The present disclosure provides methods of increasing the stability of RNA molecules (e.g., mRNAs). These methods involve modifying the mRNA to create modified mRNA. “mRNA stability,” as used herein, refers to mRNA having an increased half-life, and/or showing increased resistance to degradation through enzymatic or other regulatory means (e.g., in vivo, 20 such as in a cell), as compared to an unmodified mRNA. Accordingly, the methods herein modify mRNAs as described herein to include modifications to prevent rapid degradation by endo- and exo-nucleases, or otherwise increase half-life, as compared to unmodified versions of these mRNAs. In some embodiments, the mRNA is modified to include a nucleoside having a 2’-O-methyl modification. In some embodiments, the mRNA is modified at an internal site (i.e., 25 not a 5’ cap or 3’ end site). In some embodiments, modified nucleosides at internal sites within an mRNA are modified by using, at least in part, the activity of a 2’-O-methyltransferase polypeptide, or a fragment, analog, or variant thereof. In some embodiments, the 2’-O-methyltransferase polypeptide is selected from the group consisting of cap methyltransferase 1 (CMTR1), cap30 methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O- methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA 92 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 methyltransferase 11 homolog (TRMT11), tRNA methyltransferase 13 homolog (TRMT13), or tRNA methyltransferase 44 homolog (TRMT44). In some embodiments, the method involves identifying one or more miRNA binding sites within an mRNA and targeting the identified miRNA sites for modification using a 2’-O-methyltransferase. 2’-O-methyltransferases useful in 5 embodiments of the present disclosure are discussed in more detail, for example, in Dimitrova et al., Genes (2019) 10:117, the contents of which are incorporated herein by reference. In some embodiments, mRNAs including 2’-O-methyl modifications to nucleosides at internal sites within the mRNA are created using methods such as in-vitro transcription. In-vitro transcription to produce modified mRNAs of the present disclosure may be performed using 10 RNA polymerases. In some embodiments, the RNA polymerases are T7 polymerases. In some embodiments, the RNA polymerases include thermostabilizing mutations. In some embodiments, thermostabilized mutants of T7 RNA polymerase include one or more mutations selected from the group consisting of: R425 (e.g., R425C), G542 (e.g., G542V), Y639 (e.g., Y639G, Y639F, Y639V), H784 (e.g., H784A, H784G, H784S), E593G, V685A, I119V, G225S, 15 K333N, D366N, F400L, S661G, F880Y, H772R, S430P, N433T, S633P, F849I, P266L. Mutant T7 RNA polymerases useful in embodiments of the present disclosure are described further, for example, in Meyer et al., Nucleic Acids Research (2015) 43(15):7480-7488, the contents of which are hereby incorporated by reference. The use of in-vitro transcription to create modified RNAs including 2’-O-methyl modifications at internal sites is described further, for example, in 20 Cavaillé et al., Nature (1996) 383:732-735, Zhao and Yu, Nature Methods (2008) 5(1):95-100, Ge et al., RNA (2010) 16:1078-1085, and Elliott et al., Nature Communications (2019) 10:3401, the contents of each of which are hereby incorporated by reference. In the context of rRNA, the addition of 2'-O-Me is carried out by the protein complex known as box C/D small nucleolar ribonucleoproteins (Box C/D snoRNPs), which consist of the 25 proteins fibrillarin (FBL), SNU13, NOP56, and NOP58. Each complex further contains a snoRNA belonging to the C/D box family and are predominantly localized in the nucleolus, a subnuclear compartment dedicated to rRNA processing. Different snoRNP complexes contain different snoRNAs. The snoRNAs act as guides, utilizing their complementary base-pairing interactions with the rRNA molecule to precisely direct the site-specific 2'-O-Me modification. 30 This intricate mechanism ensures the accurate placement of 2'-O-Me modifications at specific nucleotides within the rRNA, thereby contributing to the structural and functional integrity of the ribosome and its role in protein synthesis. The enzyme FBL is a vital component of the box C/D snoRNP complex. Accordingly, in embodiments of the present disclosure, a guide 93 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 polynucleotide is complexed with the 2’-O-methyltransferase to direct site-specific modification of mRNAs. mRNAs used in methods for increasing stability of mRNAs disclosed herein may first be synthesized using and/or modified by methods well established in the art, such as those described 5 in "Current Protocols in Nucleic Acid Chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference in its entirety. In one embodiment of the aspects described herein, a template for a modified mRNA is synthesized using "splint-mediated ligation," which allows for the rapid synthesis of DNA 10 constructs by controlled concatenation of long oligos and/or dsDNA PCR products and without the need to introduce restriction sites at the joining regions. It can be used to add generic untranslated regions (UTRs) to the coding sequences of genes during T7 template generation. Splint mediated ligation can also be used to add nuclear localization sequences to an open reading frame, and to make dominant-negative constructs with point mutations starting from a 15 wild-type open reading frame. Briefly, single-stranded and/or denatured dsDNA components are annealed to splint oligos which bring the desired ends into conjunction, the ends are ligated by a thermostable DNA ligase and the desired constructs amplified by PCR. A modified mRNA is then synthesized from the template using an RNA polymerase in vitro. After synthesis of a modified mRNA is complete, the DNA template is removed from the transcription reaction prior 20 to use with the methods described herein. In embodiments of the present disclosure, mRNAs modified with the methods disclosed herein (e.g., modified at an internal site with a 2’-O-methyl modification) have at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 25 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, or greater increase in half life as compared to a reference (e.g., unmodified version) mRNA. In some embodiments, mRNAs modified with the methods disclosed herein (e.g., modified at an internal site with a 2’-O-methyl modification) have about a 10%-300% increase 30 in half life, about a 50%-300% increase in half life, or about a 50%-200% increase in half life. Other mRNA Modifications In some embodiments, the modified mRNAs may include other modifications other than 2’-O-methylation (Nm). Other modifications which may be further applied to the modified 94 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 mRNAs disclosed herein include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or 5 bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. To the extent that such modifications interfere with translation (i.e., results in a reduction of 50% or more in translation relative to the lack of the modification--e.g., 10 in a rabbit reticulocyte in vitro translation assay), the modification is not suitable for the methods and compositions described herein. Specific examples of modified mRNA compositions useful with the methods described herein include, but are not limited to, RNA molecules containing modified or non-natural internucleoside linkages. Modified mRNAs having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the 15 internucleoside linkage. In other embodiments, the modified mRNA has a phosphorus atom in its internucleoside linkage(s). Non-limiting examples of modified internucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral 20 phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are 25 also included. Representative U.S. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Pat. Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 30 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39,464, each of which is herein incorporated by reference in its entirety. 95 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Modified internucleoside linkages that do not include a phosphorus atom therein have internucleoside linkages that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having 5 morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. 10 Representative U.S. patents that teach the preparation of modified oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated 15 by reference in its entirety. Some embodiments of the modified mRNAs described herein include nucleic acids with phosphorothioate internucleoside linkages and oligonucleosides with heteroatom internucleoside linkage, and in particular --CH2-NH--CH2-, --CH2-N(CH3)-O--CH2-[known as a methylene (methylimino) or MMI], --CH2-O--N(CH3)-CH2-, --CH2-N(CH3)-N(CH3)-CH2- and -- 20 N(CH3)-CH2-CH2-[wherein the native phosphodiester internucleoside linkage is represented as --O--P--O--CH2-] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240, both of which are herein incorporated by reference in their entirety. In some embodiments, the nucleic acid sequences featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No.5,034,506, herein 25 incorporated by reference in its entirety. A modified mRNA can comprise at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the oligonucleotide. At a minimum, a modified mRNA molecule comprising at least one modified nucleoside comprises a single nucleoside with a modification as 30 described herein. Modified mRNAs of the present disclosure include at least one modified nucleoside having a 2’-O-methyl modification (Nm). It is not necessary for all positions in a given modified mRNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single modified mRNA or even at a single nucleoside within a modified mRNA. However, it is preferred, but not absolutely 96 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 necessary, that each occurrence of a given nucleoside in a molecule is modified (e.g., each cytosine is a modified cytosine e.g., 5mC). However, it is also contemplated that different occurrences of the same nucleoside can be modified in a different way in a given modified mRNA molecule (e.g., some cytosines modified as 5mC, others modified as 2'-O-methylcytidine 5 or other cytosine analog). The modifications need not be the same for each of a plurality of modified nucleosides in modified mRNA. A modified mRNA can also contain a mixture of both modified and unmodified nucleosides. As used herein, "unmodified" or "natural" nucleosides or nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and 10 uracil (U). Further modified nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Int. Appl. No. PCT/US09/038,425, filed Mar.26, 2009; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 15 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,30; 5,134,066; 20 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference in its entirety, and U.S. Pat. No.5,750,692, also herein incorporated by reference in its entirety. 25 The modified mRNAs described herein can further comprise a 5' cap. In some embodiments of the aspects described herein, the modified mRNAs comprise a 5' cap comprising a modified guanine nucleotide that is linked to the 5' end of an RNA molecule using a 5'-5' triphosphate linkage. As used herein, the term "5' cap" is also intended to encompass other 5' cap analogs including, e.g., 5' diguanosine cap, tetraphosphate cap analogs having a methylene- 30 bis(phosphonate) moiety (see e.g., Rydzik, A M et al., (2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having a phosphorothioate modification (see e.g., Kowalska, J. et al., (2008) RNA 14(6):1119-1131), cap analogs having a sulfur substitution for a non-bridging oxygen (see e.g., Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755), N7-benzylated dinucleoside tetraphosphate analogs (see e.g., Grudzien, E. et al., (2004) RNA 10(9):1479-1487), 97 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 or anti-reverse cap analogs (see e.g., Jemielity, J. et al., (2003) RNA 9(9): 1108-1122 and Stepinski, J. et al., (2001) RNA 7(10):1486-1495). In one such embodiment, the 5' cap analog is a 5' diguanosine cap. In some embodiments, the synthetic, modified RNA does not comprise a 5' triphosphate. 5 The 5' cap is important for recognition and attachment of an mRNA to a ribosome to initiate translation. The 5' cap also protects the modified mRNA from 5' exonuclease mediated degradation. It is not an absolute requirement that a modified mRNA comprise a 5' cap, and thus in other embodiments the modified mRNAs lack a 5' cap. However, due to the longer half-life of modified mRNAs comprising a 5' cap and the increased efficiency of translation, modified 10 mRNAs comprising a 5' cap are preferred herein. The modified mRNAs described herein can further comprise a 5' and/or 3' untranslated region (UTR). Untranslated regions are regions of the RNA before the start codon (5') and after the stop codon (3'), and are therefore not translated by the translation machinery. Modification of an RNA molecule with one or more untranslated regions can improve the stability of an mRNA, 15 since the untranslated regions can interfere with ribonucleases and other proteins involved in RNA degradation. In addition, modification of an RNA with a 5' and/or 3' untranslated region can enhance translational efficiency by binding proteins that alter ribosome binding to an mRNA. Modification of an RNA with a 3' UTR can be used to maintain a cytoplasmic localization of the RNA, permitting translation to occur in the cytoplasm of the cell. In one 20 embodiment, the modified mRNAs described herein do not comprise a 5' or 3' UTR. In another embodiment, the modified mRNAs comprise either a 5' or 3' UTR. In another embodiment, the modified mRNAs described herein comprise both a 5' and a 3'UTR. In one embodiment, the 5' and/or 3' UTR is selected from an mRNA known to have high stability in the cell (e.g., a murine alpha-globin 3' UTR). In some embodiments, the 5' UTR, the 3' UTR, or both comprise one or 25 more modified nucleosides. In some embodiments, the modified mRNAs described herein further comprise a Kozak sequence. The "Kozak sequence" refers to a sequence on eukaryotic mRNA having the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another `G`. The Kozak consensus sequence is 30 recognized by the ribosome to initiate translation of a polypeptide. Typically, initiation occurs at the first AUG codon encountered by the translation machinery that is proximal to the 5' end of the transcript. However, in some cases, this AUG codon can be bypassed in a process called leaky scanning The presence of a Kozak sequence near the AUG codon will strengthen that codon as the initiating site of translation, such that translation of the correct polypeptide occurs. 98 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Furthermore, addition of a Kozak sequence to a modified mRNA will promote more efficient translation, even if there is no ambiguity regarding the start codon. Thus, in some embodiments, the modified mRNAs described herein further comprise a Kozak consensus sequence at the desired site for initiation of translation to produce the correct length polypeptide. In some such 5 embodiments, the Kozak sequence comprises one or more modified nucleosides. In some embodiments, the modified mRNAs described herein further comprise a "poly (A) tail", which refers to a 3' homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides). The inclusion of a 3' poly(A) tail can protect the modified mRNA from degradation 10 in the cell, and also facilitates extra-nuclear localization to enhance translation efficiency. In some embodiments, the poly(A) tail comprises between 1 and 500 adenine nucleotides; in other embodiments the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 15 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 adenine nucleotides or more. In one embodiment, the poly(A) tail comprises between 1 and 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises between 90 and 120 adenine nucleotides. In some such embodiments, the poly(A) tail comprises one or more modified nucleosides. 20 It is contemplated that one or more modifications to the modified mRNAs described herein permit greater stability of the modified mRNA in a cell or tissue in vivo. To the extent that such modifications permit translation and either reduce or do not exacerbate a cell's innate immune or interferon response to the modified mRNA with the modification, such modifications are specifically contemplated for use herein. Generally, the greater the stability of a modified 25 mRNA, the more protein can be produced from that modified mRNA. Typically, the presence of AU-rich regions in mammalian mRNAs tend to destabilize transcripts, as cellular proteins are recruited to AU-rich regions to stimulate removal of the poly(A) tail of the transcript. Loss of a poly(A) tail of a modified mRNA can result in increased RNA degradation. Thus, in one embodiment, a modified mRNA as described herein does not comprise an AU-rich region. In 30 particular, it is preferred that the 3' UTR substantially lacks AUUUA sequence elements. Oligonucleotide Synthesis and Purification In some embodiments, the modified mRNA can be chemically synthesized using methods described herein. RNA may be produced enzymatically or by partial/total organic 99 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. In one embodiment, each strand is prepared chemically. Methods of synthesizing RNA molecules are known in the art, in particular, the chemical synthesis methods as described in Verma and Eckstein (1998) or as described herein. Generally, modified mRNA molecules can 5 by synthesized using solid phase oligonucleotide synthesis methods as described in, for example, Usman et al., U.S. Pat. Nos.5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086. In a non-limiting example, modified mRNA molecules comprising one or more modified nucleotides are synthesized, using solid phase phosphoramidite chemistry, deprotected and 10 desalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie, 1993; Wincott et al., 1995). The oligomers may be purified using ion-exchange high performance liquid chromatography (IE-HPLC), for example, on an Amersham Source 15Q column (1.0 cm.times.25 cm) (Amersham Pharmacia Biotech, Piscataway, N.J.) using a step-linear gradient. Samples are monitored at 260 nm and peaks 15 corresponding to the full-length oligonucleotide species are collected, pooled, desalted, and lyophilized. The purity of each synthesized oligonucleotide is determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.). Relative molecular masses of oligomers can be obtained, often within 0.2% of expected molecular mass. 20 The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal 25 Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the 30 invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and 100 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. 101 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 EXAMPLES Example 1: Nm at mRNA internal sites is positively associated with mRNA stability 5 To investigate the association between Nm and mRNA stability (FIG.1A), an integrative analysis of multiple public datasets was performed. To this end, mRNA half-life data was collected, as determined by RNA-seq following Actinomycin D (ActD) inhibition of transcription in HEK293T cells (Murakawa, et al. (2015Nat Commun 6, 7367. 10.1038/ncomms8367). mRNA Nm data was also collected, as determined by Nm-seq in 10 HEK293T cells(Dai et al., Nat Methods 14, 695-698). Gene Set Enrichment Analysis (GSEA) indicated that 2′-O-methylated mRNAs were enriched in mRNAs with a long half-life (FIG.1B). Compared to mRNAs that showed no detectable Nm modification, the half-lives of 2′-O-methylated mRNAs were significantly longer (FIG.1C). Because FBL is one of the enzymes well known to catalyze Nm and NOP56 is 15 required to facilitate the reaction, new RNA-Seq was performed following ActD inhibition of transcription to profile RNA half-life in control, FBL-knockdown and NOP56-knockdown HEK293T cells (FIGs.1D, 8A-8C). When compared to mRNAs that have no detectable Nm in the control cells, the mRNAs that were 2′-O-methylated in the control cells were significantly more likely to show a decrease of mRNA half-life in response to the knockdown of FBL or20 NOP56 (FIGs.1E-1F). For the mRNAs 2′-O-methylated in control cells, the reduction of half- lives was significant both upon FBL knockdown and upon NOP56 knockdown (FIGs.1G-1H). These data indicated that the Nm modifications at mRNA internal sites are positively associated with greater stability of the mRNAs. 25 Example 2: The binding of FBL on mRNA is positively associated with mRNA stability and required for mRNA Nm modification A PAR-CLIP-seq dataset from HEK293T cells (Kishore, et al., (2013). Genome Biol 14, R45.10.1186/gb-2013-14-5-r45) was analyzed. Consistent with a previous report (Dai et al., Nat Methods 14, 695-698.), FBL-binding mRNAs were found to overlap significantly with 2′-O- 30 methylated mRNAs (FIGs.2A, 8D). About 60% of the Nm-modified transcripts showed the binding of FBL. Concerning the unmethylated transcripts, only approximately 10% could be bound by FBL (FIG.2A). The genes of FBL-binding mRNAs (Kishore et al., (2022). Nucleic Acids Res 50, D287-D294.10.1093/nar/gkab702) from HEK293T cells were next used together with the mRNA half-life data (Murakawa et al., Nat Commun 6, 7367) to determine whether the 102 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 binding of FBL was required to regulate the mRNA stability. Like the mRNAs with Nm (FIGs. 1C, 8E), the FBL-binding mRNAs showed significantly longer mRNA half-life when compared to the mRNAs showing no detectable binding of FBL (FIGs.2B, 8F). Among FBL-binding mRNAs, the mRNAs that displayed a higher FBL-binding intensity appeared to be more stable 5 (FIGs.2C, 8G), suggesting that the binding intensity of FBL was positively associated with the stabilization of mRNA in the cells. To investigate whether the enhanced stability of mRNAs relies on FBL, the new RNA-seq data was further analyzed to determine the half-life change of FBL-binding mRNA in response to the knockdown of FBL and its partner protein NOP56. It was observed that the FBL-binding mRNAs were significantly more likely to show a decrease in 10 half-life compared to the mRNAs that showed no detectable binding of FBL in the wild-type cells (FIGs.2D-2E). The reduction in the half-life of FBL-binding mRNAs was significant in response to FBL knockdown and NOP56 knockdown (FIGs.2F, 8H). To determine if FBL is required for Nm modification on mRNAs, the RTL-P assay (Dong et al., (2012). Nucleic Acids Res 40, e157.10.1093/nar/gks698), a sensitive method 15 widely used, was used to confirm that ACTG1, PGHDH, SNHG5, and CALR are bound by FBL and Nm modified. It was observed that knockdown of FBL and NOP56 both increased the reverse transcription products at the low dNTP concentration condition but not at the high dNTP concentration condition (FIG.2G). Quantitative analysis of the methylation ratio revealed a significant decrease in Nm levels on all four mRNAs following the knockdown of FBL and 20 NOP56 (FIG.2H). The RNA-seq following inhibition of transcription by ActD demonstrated a decrease of half-life and expression levels of these four mRNAs upon the FBL knockdown and NOP56 knockdown (FIGs.2I-2J, 8H). Together, this verified that the expression of FBL was important for mRNA Nm modification and, thus, to stabilize these mRNAs. 25 Example 3: Upregulation of FBL is associated with increased stability and expression levels of FBL-binding mRNAs in cancer cells Analysis of TCGA patient data (Chandrashekaret al.,(2022). Neoplasia 25, 18-27. 10.1016/j.neo.2022.01.001) confirmed that FBL, as well as its partner proteins NOP56 and NOP58, were up-regulated in most of the cancer types (FIGs.9A-9C), including prostate cancer 30 (PCa) (FIG.10A). To further understand the role of FBL in cancer cells, the prostate primary epithelial cell PrEC was compared with PCa cell line C4-2 (Gao et al., (2023). Nucleic Acids Res 51, 11534-11548.10.1093/nar/gkad838). The results indicated that the RNA expression levels of FBL, NOP56, and NOP58 were higher in C4-2 cells than in PrEC (FIG.10B). Knockdown of FBL could inhibit PCa progression by regulating Nm methylation of 103 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 rRNA and thus IRES-dependent translation of oncogenes (Marcel et al., Cancer Cell 24, 318- 330). However, it was unclear if the oncogenic role of FBL also depended on direct binding and regulation of mRNAs. To investigate this unrecognized role of FBL, RIP-seq was performed in C4-2 cells to identify FBL-binding RNAs (FIG.9D). The C4-2 cell data was compared with 5 public HEK293T cell data to verify the consistency of the key features of FBL-binding RNAs. Among the FBL-binding transcripts in C4-2 cells, 1377 transcripts were bound by FBL in both C4-2 and HEK293T cells, whereas FBL bound 3468 transcripts in C4-2 but not in HEK293T cells (FIG.9E). Consistent with previous findings in HEK293T cells (Kishore et al., Genome Biol 14, R45; Porter et al., (2021). Nat Commun 12, 1569.10.1038/s41467-021-21623-4), 10 nearly 80% of the FBL-binding RNAs in C4-2 cells were protein-coding genes (FIG.3C). The data in C4-2 also confirmed that FBL could bind to the CD-box snoRNAs and mRNAs (FIGs. 3C and 9F-9H). Therefore, consistent key features of FBL-binding RNAs were observed in the new data from C4-2 cells and public data from HEK293T cells. It was next investigated if FBL regulates the expression of FBL-binding mRNAs in the 15 C4-2 cells. FBL-binding mRNAs were observed to be significantly enriched with the mRNAs down-regulated upon FBL knockdown (FIG.3D). Compared to mRNAs that showed no detectable binding of FBL, the FBL-binding mRNAs were significantly more likely to have decreased RNA expression levels in response to the knockdown of FBL (FIG.3E). Moreover, the FBL-binding transcripts were more likely to show a decrease in half-life than those with no 20 detectable FBL binding (FIG.3F). The half-lives of FBL-binding mRNAs appeared to be significantly shorter in the FBL knockdown cells compared to the control (FIG.3G). Also, compared to mRNAs that had no binding of FBL in C4-2 cells, the FBL-binding mRNAs in C4- 2 cells were more likely up-regulated in C4-2 cells when compared to PrEC (FIG.3H). The genes of FBL-binding mRNAs in C4-2 cells showed higher expression levels in C4-2 cells 25 compared to PrEC cells (FIGs.3I-3J). These data suggested that the binding of FBL on mRNAs upregulated the mRNA expression level by increasing the stability of these mRNAs in the C4-2 cells. Example 4: A machine learning model to detect Nm sites in mRNAs by Nanopore 30 sequencing Until now, Nm at mRNA internal sites has not yet been studied in cancer. This is partly due to the lack of a robust technique to measure Nm stoichiometry across the transcriptome quantitatively. To fill this gap, Nanopore direct RNA-seq we adapted with machine learning techniques to detect Nm modifications with stoichiometry on mRNAs. Recently, the XGBoost 104 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 machine learning method was successfully applied to detect RNA modifications such as m6A (Gao et al., Genome Biol 22, 22) and Nm (Salem et al., bioRxiv). However there is no algorithm to detect de novo and measure the stoichiometry of Nm on mRNA. The XGBoost machine learning method was customized to use reported rRNA Nm sites as the training data, as these 5 sites have a high Nm ratio compared to a low Nm ratio at sites on mRNA. Also, rRNA Nm sites have been very well studied using riboMeth-seq (Birkedal et al., (2015). Angew Chem Int Ed Engl 54, 451-455.10.1002/anie.201408362; Marchand et al., (2016). Nucleic Acids Res 44, e135.10.1093/nar/gkw547; Gumienny et al., (2017). Nucleic Acids Res 45, 2341-2353. 10.1093/nar/gkw1321), 2′-OMe-Seq (Zhu et al., (2017). RNA 23, 1303-1314. 10 10.1261/rna.061549.117), and RibOxi-Seq (Incarnato et al., (2017). Nucleic Acids Res 45, 1433-1441.10.1093/nar/gkw810), whereas mRNA Nm sites were much less studied before. The 107 well-known Nm sites reported were thus collected for 5.8S, 18S, and 28S rRNAs in humans (Pichot et al., (2020). Front Genet 11, 38.10.3389/fgene.2020.00038). After that, using the public Nanopore direct RNA-seq dataset for rRNAs as positive data and in vitro-transcribed 15 (IVT) RNAs without any modification as negative data, an XGBoost machine-learning model was trained for the 107 sites containing one hundred 5-mers in the rRNAs (FIGs.4A, 10A). For these 5-mers, the area under the receiver operating characteristic (AUROC) and model performance accuracy ranged from 0.98 to 0.99 and from 0.92 to 0.94, respectively (FIGs.4B, 10B-10C). This result indicated that the new machine-learning model accurately recaptured the 20 known rRNA Nm sites. This machine learning model was then applied to the Nanopore direct RNA-seq datasets generated for control and FBL-deficient C4-2 cells (FIG.10D). The model successfully recaptured the reported Nm sites of rRNA (FIGs.4C-4D). None of the predicted Nm sites overlapped with the 103 other known types of modification on rRNA (Pichot et al., Front Genet 25 11, 38), i.e., the 91 Psi sites, a m7G sites, two m6A sites, two m62A sites, a m1A sites, a m1Acp3P sites, two Ac4C sites, two m5C sites, and a m3U site (FIG.10E). Notably, the Nm ratio at detected Nm sites appeared significantly reduced by the knockdown of FBL (FIGs.4E- 4F). The machine learning model was also applied to measure the rRNA modification ratios in a wild-type yeast strain and three other strains, each with knockout of snoRNAs: snR60, snR61, 30 and snR62. The model recaptured the reported snoRNA-specific deficiency of Nm modification in each snoRNA knockout strain (FIG.4G). Moreover, the method revealed a marked decrease in Nm ratios within the ribosome RNAs of Drosophila cells upon FBL knockdown (FIGs.4H- 4I). These results indicated that by combining XGBoost machine learning and Nanopore direct RNA-seq techniques, the stoichiometric change of known Nm sites in rRNA was successfully 105 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 detected with a base resolution. This model was then used to identify Nm modifications on each of the 1005-mers throughout all mRNAs in the C4-2 cells. The model identified 26,170 Nm sites for mRNAs of 4,586 genes. The methylation ratio at these Nm sites of mRNAs ranged from 0.1 to 1, with an 5 average ratio of 0.30. In contrast, the methylation ratio of Nm sites in rRNA is three-fold higher, with an average ratio of 0.92 (FIG.4J). This observation is reminiscent of an early report that rRNAs have a much longer half-life than mRNA (Abelson et al., (1974). Cell 1, 161-165) and the new observation that Nm is positively associated with mRNA stability. The base G is preferentially deposited with Nm, with 40% Nm being on G (FIG.10F), whereas only 24% of 10 bases are G in the mRNA, and 29% of bases are G in the 1005-mers covered by the machine learning model (FIG.10F). Most of these Nm sites on mRNA were in the exon and 3′ UTR regions (FIGs.4K, 10G). Above all, the machine learning model successfully recaptured well- known rRNA Nm sites and revealed many new ones with stoichiometry on mRNAs based on Nanopore direct RNA-seq data. 15 Example 5: FBL promotes widespread Nm on mRNAs with increased RNA stability in PCa cells It was next investigated whether Nm modification at mRNA internal sites in cancer cells was associated with an elevated expression of mRNAs in cancer pathways. To test if FBL 20 regulated the Nm modification on mRNAs in cancer cells, FBL was knocked down in C4-2 cells and the effect on mRNA Nm modification was analyzed, based on Nanopore direct RNA-seq. The overall methylation ratios calculated by the machine learning model at mRNA Nm sites across the transcriptome were significantly decreased upon the FBL suppression (FIG.5A). KEGG pathway analysis showed that 2′-O-methylated transcripts in C4-2 cells were enriched in 25 PCa-related pathways (FIG.11A). These 2′-O-methylated mRNAs were enriched with the mRNAs showing higher expression levels in C4-2 cells relative to PrEC (FIG.11B). Compared to mRNAs that showed no detectable Nm in C4-2 cells, the 2′-O-methylated mRNAs were more likely to be up-regulated in C4-2 cells relative to PrEC (FIGs.11C-11D). These data indicated that Nm modification was enriched on cancer-related mRNAs upregulated in PCa cells relative 30 to normal cells. Differential methylation analysis was conducted at individual sites and it was found that 1568 sites were hypo-methylated upon FBL knockdown, while only 285 sites were hyper- methylated (FIG.5B). The average density plot of Nm sites indicated that the decrease of methylated sites happened in all regions of the mRNA sequence (FIG.5C). This pattern 106 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 remained similar when the analysis was repeated for each of the four RNA bases (FIG.10G). Further, a significant overlap between FBL-binding mRNAs and 2′-O-methylated mRNAs was observed in the C4-2 cells (FIG.5D). These results suggested that FBL is required for thousands of Nm sites on mRNAs in C4-2 cells. 5 To further understand the role of Nm modification in FBL-mediated mRNA expression regulation, the association between mRNA expression changes and Nm changes in response to FBL knockdown in C4-2 cells was analyzed. The genes with higher mRNA expression levels showed higher mRNA Nm ratios (FIG.5E), consistent with a previous report in HEK293T and Hela cells (Dai et al., Nat Methods 14, 695-698). Three gene groups were next defined in C4-2 10 cells, including genes showing hypo-methylated mRNAs upon knockdown of FBL, all genes showing Nm modification on mRNAs, and genes showing no detectable Nm on mRNAs. The genes showing Nm on mRNAs tended to be downregulated by FBL knockdown (FIGs.5F-5G). The mRNA half-life among the three gene groups was next compared and it was found that the genes with hypo-methylated mRNAs had the highest RNA stability, whereas the genes with no 15 detectable Nm on mRNAs had the shortest RNA half-life (FIG.5H). Furthermore, upon the knockdown of FBL, the genes with hypo-methylated mRNAs showed the most significant decrease in mRNA half-life, whereas the genes with no detectable Nm on mRNAs showed the most minor decrease in half-life (FIGs.5I-5J). These results were further confirmed by profiling RNA half-life using the SLAM-seq method, which does not 20 require the inhibition of transcription (FIGs.11E-11M). In addition, the 2′-O-methylated mRNAs were also defined based on Nanopore direct RNA-seq data in HEK293T cells and similar results as in the C4-2 cells were observed, i.e., the 2′-O-methylated mRNAs overlapped significantly with FBL-binding mRNAs (FIG.11N), showed longer half-life when compared to mRNAs with no detectable Nm (FIGs.11O-11Q), and were more likely to show decreased half- 25 life upon FBL knockdown and NOP56 knockdown (FIG.11R). Intriguingly, the stability of the 2'-O-methylated long noncoding RNAs, compared to that of unmethylated long non-coding RNAs, also appeared to be more significant and more sensitive to FBL knockdown (FIG.5K- M). To exclude the potential confounding effect of translation efficiency and gene expression 30 level, mRNA half-life analysis was performed between two gene groups specifically selected to have similar translational efficiency change upon FBL knockdown (FIGs.12A-12F) or expression level (FIGs.12L-12P), but with one group bearing Nm and the other group showing no detectable Nm. The Nm group was found to be still more stable than the unmodified mRNAs. The distributions of Nm sites at the different codon positions were checked in both C4-2 (FIG. 107 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 12G) and HEK293T (FIG.12J) cells. Interestingly, the number of genes with Nm at the 2nd codon position was less than those with Nm at the 1st and 3rd positions. As previous studies showed that Nm at the 2nd position hinders the translation (Hoernes et al.,Nucleic Acids Res 44, 852-862.10.1093/nar/gkv1182;), it is possible that Nm at the 2nd position was negatively 5 selected by evolutionary pressure or translational stress. Moreover, mRNAs with Nm at the second codon position were less stable than those with Nm at the two other codons in the C4-2 (FIGs.12H-12I) and HEK293T cells (FIG.12K). This observation is in accordance with the report that Nm at the 2nd codon position hindered translation (Hoernes et al., Genes (Basel) 10), and impaired translation promoted RNA decay 10 (Presnyak et al., (2015). Cell 160, 1111-1124.10.1016/j.cell.2015.02.029;). The paired Wilcoxon test was used to compare the RNA decay rates between the siCTRL and siFBL samples. By requiring a p-value smaller than 0.05, 3,176 and 236 genes were detected with decreased and increased RNA half-life upon FBL knockdown, respectively (FIG.12Q). Among them, 33% of the genes showing a decreased RNA half-life were 2'-O-methylated in mRNA, 15 which is a 1.81-fold enrichment compared to random expectation (FIG.12R-12S). A significant overlap between Nm sites detected by the Nanopore method and the Nm-Mut-seq methods (Chen et al., Cell Res 33, 727-730) was observed, with the number of overlapped sites 40 folds greater than expected by chance (Fisher’s exact test P value < 1e-16) (FIGs.12T-12U). Meanwhile, no significant overlap was observed between the Nm sites and sites of other 20 detectable modification types on mRNAs, with the number of overlapped sites always smaller than expected by chance (FIG.12V). Together, these data suggested that for most of the Nm sites, their influence on RNA stability was not a confounding effect of RNA expression level, translation efficiency, or other RNA modification types. 25 Example 6: FBL-mediated Nm in mRNA is required for the cancer phenotypes of C4-2 cells Next, RTL-P experiments were performed to validate 10 hyper-methylated sites of eight genes in C4-2 cells (FIG.6A). The results confirmed that at least 7 sites showed a decrease of Nm in response to FBL-knockdown in C4-2 cells (FIGs.6B-6C). A reduction in mRNA 30 expression level was also observed for these genes upon the FBL knockdown (FIG.6D). Importantly, these 2′-O-methylated mRNAs also showed increased expression in PCa samples compared to normal samples in the TCGA patient datasets (FIG.6E). To investigate whether differences in Nm levels in specific mRNAs regulate tumor progression, a focal study was performed on the gene PSMD13 (FIG.6E and FIG.12W). A 108 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 decrease in PSMD13 protein level was observed following FBL knockdown, which could be rescued by the re-expression of the wild-type, but not a catalytically dead mutant of FBL, a four- point mutant of FBLT172A, D191A, F192A, D216A (Zhou et al. (2023). Cancer Discov 13, 332-347.10.1158/2159-8290.CD-22-0210) (referred as FBL-mu) (FIG.6F). Consistently, 5 decreases in Nm on PSMD13 mRNA upon FBL knockdown were verified and could be rescued by the overexpression of the wild-type FBL but not the mutant of FBL (FIG.6G-6H). In addition, elevated expression of PSMD13 was associated with PCa progression (FIG.6I) and poor patient survival in the TCGA cohort (Chandrashekar et al., Neoplasia 25, 18-27;) (FIG.6J). Knockdown of PSMD13 decreased cancer cell proliferation, invasion, and migration (FIG.6K- 10 6P). These data supported the hypothesis that FBL catalyzed Nm on mRNA to increase the expression of PSMD13 and thus played an oncogenic role in an Nm-dependent manner. Example 7: RNA stabilization by Nm was associated with 3′ UTR shortening To investigate the mechanism by which Nm regulated mRNA stability, a comprehensive 15 analysis was performed to explore mRNA sequence features associated with Nm in C4-2 (FIGs. 7A-7C, 13A-13H) and HEK293T cells (FIGs.13K-13W). These features included 3′ UTR length, miRNA binding sites, ARE element, exon length, intron length, and GC content. For instance, the genes of hypo-methylated transcripts, upon knockdown of FBL (Hypo) and 2′-O- methylated transcripts (Nm) in the control condition were found to both show shorter 3′ UTR 20 lengths compared to the unmethylated transcripts in C4-2 (FIG.7A) and HEK293T cells (FIG. 13K). Previous studies demonstrated that the length of 3′UTR was negatively correlated with RNA half-life in mice (Freimer et al., . (2018). Elife 7.10.7554/eLife.38014) and zebrafish (Mishima, Y., and Tomari, Y. (2016). Mol Cell 61, 874-885.10.1016/j.molcel.2016.02.027). The mRNAs with longer 3′UTR tended to have less stability due to an increased chance of being 25 targeted by miRNA (Mayr, C., and Bartel, D.P. (2009). Cell 138, 673-684. 10.1016/j.cell.2009.06.016) and some sequence features, such as the AU-rich elements (ARE) in the 3′ UTR (Chen, C.Y., and Shyu, A.B. (1995). Trends Biochem Sci 20, 465-470. 10.1016/s0968-0004(00)89102-1). Consistent with these reports, the methylated transcripts were found to have fewer miRNA target sites and lower ARE scores in C4-2 (FIGs.7B-7C) and 30 HEK293T cells (FIGs.13L-13M). It was next explored whether the change of FBL-mediated Nm could cause the change of 3′ UTR length of transcripts derived from the same gene. Alternative polyadenylation (APA) is an RNA-processing mechanism that could lead to different 3′ UTR lengths of transcripts derived from the same gene. To detect APA events upon FBL depletion, in-depth RNA-seq was 109 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 performed in control and FBL-knockdown cells. It was found that the FBL knockdown induced a transcriptome-wide increase of distal poly(A) site usage, which suggested a global lengthening of 3′ UTRs (FIG.7D). Genes with lengthened 3′ UTRs were significantly enriched with the genes of 2’-O-methylated transcripts, while genes with shortened 3′ UTRs showed no significant 5 enrichment or depletion of genes of unmethylated transcripts (FIGs.7E-7F, 13I). Additionally, the mRNA Nm level was higher for genes with lengthened 3′ UTRs than for genes with shortened UTRs (FIG.7G). Upon FBL knockdown, 3′ UTR lengthening was associated with a significant decrease of Nm, whereas 3′ UTR shortening showed no significant change in Nm (FIG.7G). Furthermore, the reduction of mRNA stability upon FBL knockdown was 10 significantly greater for genes with shortened 3′ UTR than for genes with lengthened 3′ UTR (FIG.7H). For instance, hypo-methylation sites, 3′ UTR lengthening, and decreased stability of mRNAs were observed for the genes AKAP8L, PSMD13, CDC6, and NQO2. (FIGs.7H-7I, 13J). Based on the findings above, it was hypothesized that specific RNA binding proteins 15 (RBPs) might interact with or recognize Nm-modified transcripts, thereby regulating APA. To test this hypothesis, the CLIPdb database of POSTAR3 (Zhao et al., Nucleic Acids Res 50, D287-D294) was used to identify RBP-binding regions colocalized with Nm sites regions in HEK293 or HEK293T cells. This analysis revealed a list of 60 RBPs, each with a significant overlap (Fisher’s exact test P value <0.01, odds ratio > 1.5) between their binding regions and 20 the Nm sites in mRNAs. The top RBPs in this list included the ATXN2 (Yokoshi et al., (2014). Mol Cell 55, 186-198.10.1016/j.molcel.2014.05.022), each with binding regions encompassing more than 10% of the Nm sites (FIGs.7J, 14A-14B), that had previously been reported to involve in regulation of RNA stability or APA. Among them, CPSF7 was reported to promote proximal APA and shorter UTRs (Tseng et al., Nucleic Acids Res 50, 9397-9412). A strong 25 positive correlation was observed between differential APA induced by the depletion of CPSF7 and FBL (FIGs.7K, 14D), while the depletion of CPSF7 and FBL did not affect the expression of each other (Consortium, E.P. (2012). An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57-74.10.1038/nature11247) (FIG. S7E). Moreover, the knockdown of FBL impaired the half-life of CPSF7-binding mRNAs (FIG.14C) and the 30 binding of CPSF7 on the Nm-modified transcripts PSMD13, CDC6, AXAPL8 and NQO2 (FIGs.7L-7M), suggesting that CPSF7 was a potential candidate affecting APA by binding 2′- O-methylated transcripts. A simple working model in which the 2′-O-methylated transcripts tend to have shorter 3′ UTR and thus reduced chance of decay mediated by mechanisms such as miRNAs and AU-rich elements was created (FIG.7M). 110 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Investigating the effect of RNA modification on RNA stability is important for understanding post-transcriptional regulation in disease (Boo, S.H., and Kim, Y.K. (2020). Exp Mol Med 52, 400-408.10.1038/s12276-020-0407-z) and developing RNA-based therapeutics (Adachi, et al.,(2021). Biomedicines 9.10.3390/biomedicines9050550). Previous studies 5 revealed that Nm in the cap of mRNA, 3′-end of siRNA and miRNAs in plants, piRNAs (Piwi- interacting RNAs) in animals protect the RNAs from degradation (Dimitrova, D.G., Teysset, L., and Carre, C. (2019). Genes (Basel) 10.10.3390/genes10020117). In addition, it is reported that 2′-O-methylation stabilizes specific RNA conformation in vitro (Abou et al.,(2020). Nucleic Acids Res 48, 12365-12379.10.1093/nar/gkaa928). However, the function of Nm at mRNA 10 internal sites was unclear until now. In the above Examples, it was shown that the internal Nm sites in mRNA regulate cellular mRNA stability. Transcriptome-wide investigations were performed, demonstrating that Nm and FBL-binding transcripts had higher mRNA stability. Taking advantage of the Nanopore sequencing technology along with the machine learning strategy, Nm sites and the dynamic changes of Nm in mRNAs across the transcriptome were 15 detected. By transcriptome-wide profiling of Nm modifications and mRNA half-life in PCa cells, it was revealed that elevation of FBL-mediated Nm modifications was associated with increased stability and expression of mRNAs in PCa pathways. Therefore, these finding suggested a new post-transcriptional role of FBL in cancer. To provide insights into the mechanism in which Nm stabilizes mRNA, the mRNA 20 sequence features were comprehensively analyzed, such as UTR length, miRNA target site enrichment, and abundance of AU-rich elements. It was found that 2′-O-methylated transcripts had shorter 3′ UTRs when compared to unmethylated transcripts. Additionally, the findings showed that knockdown of FBL increased the length of 3′ UTR, and 3′ UTR lengthening is known to decrease mRNA stability (Mishima et al., Mol Cell 61, 874-885; Mayr et al., Cell 138, 25 673-684). Besides APA, several factors, including the GC content, length of the 5' -UTR, the CDS, and translation efficiency, can also influence RNA stability. For example, it is reported that GC content is positively correlated with mRNA half-life (Kristoffersenet al., (2012). Genome Biol 13, R30.10.1186/gb-2012-13-4-r30). Moreover, the length of mRNA and GC content was reported to be associated with their subcellular localization. mRNA accumulation in 30 P-bodies is anti-correlated strongly with the GC content of their CDS and 3′ UTR (Courel, et al. (2019). Elife 8.10.7554/eLife.49708). Furthermore, low-GC content of mRNA tends to be in the P-body and be targeted by miRNAs (decay), while high-GC mRNAs could be targeted by RNA binding proteins like YTHDF2 and SMG6 (stable) (Courel et al., Elife 8). These findings revealed that Nm-modified transcripts differed from unmodified transcripts regarding GC 111 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 content and mRNA length. METHODS 5 TABLE 1: KEY RESOURCES TABLE REAGENT or RESOURCE IDENTIFIER 112 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Raw data of gels and blots https://doi.org/10.17632/yshp7typsf.1 113 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 clusterProfiler (v4.0.5) https://bioconductor.org/packages/rele EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS The human PCa cell line C4-2 was a gift from Dr. Leland W. Chung at Cedars-Sinai Medical Center and was cultured in RPMI 1640 medium supplemented with 10% fetal bovine 5 serum (FBS). HEK293T cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% FBS. Both cell lines were maintained at 37 °C and 5% CO2 in a humidified atmosphere and were authenticated and routinely screened for Mycoplasma. 10 Antibodies and reagents The antibodies used in the Examples are listed in the key resources table. Transfection of siRNA All the siRNAs used in the Examples were purchased from Thermo Fisher (siFBL-1: 15 s4820, siFBL-2: s4821; siNOP56-1: s20641, siNOP56-2: s20642, siPSMD13-1: s229818, siPSMD13-2: s229819). Lipofectamine RNAiMAX (Invitrogen) was utilized for siRNA transfection per the manufacturer’s protocol. Cells were collected at 3 days post-transfection for further use. If the number of siRNA was not indicated in the Examples, it should represent siRNA-1. 20 RNA extraction and Real-time (RT)- qPCR analysis Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer’s protocol. First-strand cDNA was then synthesized using the Maxima H Minus First Strand cDNA Synthesis kit (Thermo). The obtained cDNA samples were amplified using 25 Universal SYBR Green Supermix (Bio-Rad) in a QuantStudio 6 Flex Real-time PCR system (GE Healthcare) following the manufacturer’s instructions. The relative RNA level was calculated using the 2−ΔΔCt method, with the Ct values normalized using GAPDH as an internal control. 114 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Reverse Transcription at Low dNTP concentrations followed by PCR (RTL-P) method to detect Nm levels in mRNA To validate the 2′-O methylation in mRNA, total RNA was extracted from control, FBL- knockdown, and NOP56-knockdown cells using RNeasy Plus Mini Kit (Qiagen). Reverse 5 transcription (RT) was performed using specific reverse primers targeting mRNA sequences upstream to one methylation site in the presence of either a low level (1 μM) or a high level (1 mM) of dNTP. The RT reaction mixture contains 200 ng RNA, 200 U M-MLV reverse transcriptase (Promega), 10 mM RT primers, 0.5 U RNase Inhibitor (Promega), and deoxynucleotide (dNTP) (low/high, Invitrogen). The obtained cDNA was subjected to two 10 separate amplification reactions using two pairs of primers targeting upstream and downstream of the methylation site, respectively. The PCR mixture contains 10 μL amfisure PCR Master Mix (GenDEPOT), 2 μL cDNA and 10 mM PCR primers. The PCR products were then separated on 1% agarose gels and visualized by a Bio-rad imaging system. DNA band signal intensities were analyzed using ImageJ software. The methylation ratio of each group was determined from the 15 densities of PCR bands obtained using high and low dNTP concentrations. Total RNA extraction and quality assessment Total RNA was extracted using RNAprep Pure Kit (polysaccharides and polyphenolics- rich) and treated with DNase I to remove DNA. Firstly, the quality of total RNAs was detected 20 using 1% agarose electrophoresis, and no RNA degradation was observed. Then, the OD260/280 for all the samples was at 2.12–2.15 using a Nanodrop 2000 spectrophotometer (Thermo Scientific). Finally, the RIN value for all the samples was above 9.0 using the Agilent 2100 Bioanalyzer in combination with RNA Analysis Kits. 25 RNA half-life calculation After RNA-seq, the reads were mapped to the genome hg38 using tophat (version 2.1.1) (Kim et al., Genome Biol 14, R36.10.1186/gb-2013-14-4-r36) with default parameter values and UCSC-known genes as reference. Read counts of each gene were computed by htseq-count (version 0.11.2) (Anders et al., (2015). Bioinformatics 31, 166-169. 30 10.1093/bioinformatics/btu638). RNA half-life of the HEK293T cell and C4-2 cells was calculated as described before (Li, et al. (2023). Nucleic Acids Res 51, 6020-6038. 10.1093/nar/gkad300). The RNA levels were first normalized to RPKM (Reads Per Kilobase Million). To reduce the noise due to genes that showed a higher expression level after inhibition of transcription, the RPKM values were scaled based on the expression levels of the top 10 genes 115 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 which were most stable between time points, so that these genes will have no change of expression between time points after the normalization. The RNA degradation rate kdecay was defined as the mean values of ratio from RPKM at time 0 over the RPKM value at each other time point (0 hour, 6 hours, 12 hours, and 24 hours). After that the pseudo-mRNA half-life t1/2 5 was calculated as ln2/kdecay. SLAM-seq to measure mRNA half-life Following the manufacturer's protocol, the mRNA degradation kinetics was measured using SLAM-seq Kinetics Kit: Catabolic Kinetics Module (LEXOGEN). In general, control or 10 FBL-deficient C4-2 cells were cultivated in 1640 medium containing 100 μM 4-thiouridine (S4U) for a continuous 24-hour duration. Subsequently, the S4U-containing medium was exchanged by medium with an excess (10 mM) of unmodified uridine (U), and cell samples were collected at specific time points. The reference point for these time points was set at 0 hour, corresponding to the moment of S4U removal. RNA isolation was performed on all collected 15 samples by Trizol (Thermo), and the 4-thiol groups present on S4U-labeled transcripts were alkylated with iodoacteamide. The libraries were generated from 70-200 ng of input RNA using QuantSeq 3’ mRNA-Seq Library Prep Kit (LEXOGEN), followed by shipment to Admera Health for sequencing. To estimate mRNA half-lives, a published SLAM-seq protocol was followed. Initially, 20 raw reads in fastq files underwent quality checking and trimming using fastp (Chen, et al., (2018). Bioinformatics 34, i884-i890.10.1093/bioinformatics/bty560). Subsequently, these reads were processed with slamdunk (Neumann et al., (2019). BMC Bioinformatics 20, 258. 10.1186/s12859-019-2849-7). For mRNA half-life estimation, half-life values per gene were averaged over three replicates per condition, following the pipeline from 25 https://github.com/melonheader/HLEB (Loedige, et al, (2023). Mol Cell 83, 2709-2725 e2710. 10.1016/j.molcel.2023.06.021). A filtered version was also calculated that averaged only over data from those replicates where the calculated pseudo-R2 values were >0.95 (providing evidence for good model fits) and considered those values as ‘stringently’ filtered where at least 2 replicates remained after filtering and where the standard deviation of the considered half-life 30 values less than 1. 116 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Comparison of mRNA half-life of two transcript groups with similar expression levels and translation efficiency change To exclude the confounding effect of RNA expression and translation efficiency on RNA stability, the Nm transcripts and unmodified transcripts were combined into a pool. The genes 5 were ranked by their expression levels or translation efficiency changes and then segmented into 20 equal groups. From each group, an equal number of Nm-modified and unmodified mRNAs were randomly selected to form a subset of Nm transcripts and a subset of unmodified mRNAs to evaluate their half-lives as previously described (Li et al., Nucleic Acids Res 51, 6020-6038). 10 Western blot Cell lysates were prepared and denatured by adding 4× loading buffer (Bio-Rad) and incubating at 95°C for 10 minutes. Subsequently, the protein samples were separated through SDS-PAGE electrophoresis and semi-dry transferred onto nitrocellulose membranes (Bio-Rad). After blocking for 30 minutes in Tris-buffered saline-Tween 20 (TBST) containing 5% nonfat 15 milk, the membranes were subjected to immunoblotting by incubating with primary antibodies for 2 hours at room temperature. Following this, the membranes were washed and incubated with goat anti-mouse/rabbit IgG (H + L)-HRP secondary antibody (GenDEPOT) at a 1:5000 dilution for 1 hour. The signals were developed using a western ECL Substrate (Bio-Rad) and captured with 20 a Bio-Rad imaging system. A list of primary antibodies used in the Examples can be found in the STAR method. FBL rescue experiments FBL rescue experiments were conducted in C4-2 cells following Zhou et.al. Initially, 25 lentivirus expressing doxycycline-inducible siCtrl and siFBL (targeting the 3'-UTR of endogenous FBL mRNA) were used to infect C4-2 cells. These inducible knockdown cells were then utilized for FBL experiments. The empty vector, wild-type FBL, or mutant FBL, into the inducible FBL knockdown cells was delivered through lentivirus infection, followed by the induction of FBL knockdown with 100 ng/mL doxycycline for 3 days. 30 Cell Viability Assay Control or PSMD13-deficient C4-2 cells were seeded in 96-well plates at a density of 5 × 10^3 cells/well and incubated at 37°C in a humidified 5% CO2 atmosphere. At the designated time points, the culture medium was aspirated, and 100 μL of CellTiter-Glo 2.0 solution 117 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 (Promega) was added to each well. The plates were then placed on an orbital shaker at 37°C for 10 minutes to induce cell lysis, and bioluminescence was measured using a Tecan plate reader. Wound healing assay 5 Control or PSMD13-deficient C4-2 cells were cultured in 35 mm dishes with 3-well Culture-Insert (Ibidi) till grown to 80% confluency. After carefully removing the inserts, the dishes were replenished with serum-free 1640 medium and sustained in a humidified environment at 37°C. Microscopic images were captured at the 0-hour and 30-hour marks, and the extent of cell migration was quantified by ImageJ software. 10 Boyden chamber invasion assay To assess the invasiveness of C4-2 cells undergoing PSMD13 suppression, Boyden chamber assays were employed using Transwell inserts (Millipore) coated with Matrigel (Corning). In brief, the upper surface of polycarbonate membranes (8.0-μm pore size) within the 15 Transwell chambers was coated with a diluted Matrigel (1:20) in 1640 medium. Subsequently, cells (3 × 10^4) suspended in 300 μL of serum-free 1640 medium were added to the upper compartments of the chambers, while the lower compartments were filled with 800 μL of 1640 medium containing 10% FBS. After 24 hours, cells that had migrated from Matrigel to the lower surface of the filters were fixed with methanol, stained with 0.1% crystal violet (Sigma), and 20 subjected to microscopic examination. The number of invasive cells was determined as the average count from 6 randomly selected fields per filter. Nanopore direct RNA-seq library preparation and sequencing For library preparation, the total RNA was extracted from control and FBL-deficient C4- 25 2 cells using RNeasy Plus Mini Kit (Qiagen), followed by mRNA isolation using PolyATtract® mRNA Isolation System (Promega). The mRNA concentration was measured by Qubit RNA HS assay kit (ThermoFisher, Q32855), and the quality was confirmed by Agilent Bioanalyzer RNA Pico assay chip. Then, the mRNA was subjected to library construction using an Oxford Nanopore direct RNA sequencing kit (SQK-RNA002) according to the manufacturer’s manual. 30 500 ng of mRNA was first annealed, ligated to RT adapter, and then reverse-transcribed to form DNA-RNA hybrid products. Then, the RNA adapter was ligated to the products and was ready for sequencer loading. The resulting library was sequenced on an R9.4.1 flow cell (FLO- MIN106D) using a MinION sequencer, and FAST5 raw sequencing data was obtained in a real- time manner. 118 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Training the machine learning model by rRNA Nm data The Nanopore dataset of rRNA and unmodified in vitro transcripts (IVT) were downloaded from the SRA database by accession number SRP166020 (Jenjaroenpun, et al. 5 Nucleic Acids Res 49, e7). Each fast5 file was divided into single fast5 files using the software multi_to_single_fast5(version 3.1.6). Base-calling was performed using Guppy (version 3.6.1+249406c). Then, the re-squiggle algorithm in Tombo (version 1.5.1) was used to correct the raw signal based on reference transcripts. For the detection of Nm based on Nanopore direct RNA-seq, a machine-learning model was developed on top of the Nanom6A (Begik et al., Nat 10 Biotechnol 39, 1278-1291), developed initially to detect m6A. At first, the rRNA reads were mapped to the reference of human rRNA. Training features were extracted, including the median, standard deviation, mean, and dwell time of the Nanopore signal for the k-mer around each Nm site in the rRNA. On the other hand, these features of the same k-mer were also extracted from the IVT reads of mRNA. Then, an XGBoost model was trained by dividing the 15 features into the training and testing sets with a ratio of 4:1 for cross-validation. The XGBoost model was constructed using the machine learning library Scikit-learn. The receiver operating characteristic (ROC) curve and the area under the ROC (AUROC) were used to evaluate the performance of the model. 20 Detection of Nm based on Nanopore direct RNA-seq data After base-calling using guppy_basecaller (version 3.6.1), the Nanopore direct RNA-seq reads with quality values greater than 7 were aligned to the human genome version hg38 (GRCh38.p13 from the GENCODE database) using Minimap2 (version 2.22-r1101) with parameters “--secondary=no -ax splice -uf -k14”. The re-squiggle algorithm in Tombo version 25 1.5.1 was used to correct sequence errors based on reference transcripts downloaded from: https://www.gencodegenes.org/human. The data were then subjected to the machine learning model to detect Nm. A base found to be modified in at least 5 transcripts was identified as a modified Nm site. Differentially methylated sites between normal and knockdown cells were calculated by the R package REDITs (https://github.com/gxiaolab/REDITs) (Tran et al., (2020). 30 Bioinformatics 36, 2796-2804.10.1093/bioinformatics/btaa066). The average density of the Nm site across the transcript body was plotted using the Guitar R/Bioconductor package (Cui (2016). Biomed Res Int 2016, 8367534.10.1155/2016/8367534). A similar method is applied to the Nm detection of rRNA in yeast. The Nanopore direct RNA-seq data of rRNA in WT, snR60, snR61, and snR62 strains of yeast (Begik et al., Nat Biotechnol 39, 1278-1291), human HepG2 and 119 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 HeLa cell (Tavakoli, et al., (2023). Nat Commun 14, 334.10.1038/s41467-023-35858-w), and Drosophila cells (Sklias et al., (2023). Comprehensive map of ribosomal 2′-O-methylation and C/D box snoRNAs in Drosophila melanogaster&lt;/i&gt. bioRxiv) were downloaded from the ERA3183105, PRJNA777450 and PRJEB45722 of the European Nucleotide Archive (ENA) 5 database and GEO database, respectively. After base-calling, mapping, and feature extraction, our model trained with human rRNA was applied to detect the Nm in these samples. FBL RIP-seq and analysis The FBL-binding transcripts in HEK293T cells were obtained from the database of 10 CLIP-seq: http://postar.ncrnalab.org. The RIP experiment in C4-2 cells was performed using the EZ-Magna RIP kit (Millipore), following the procedure provided by the manufacturer. C4-2 cells were lysed in RIP lysis buffer and incubated with FBL antibody-magnetic beads mixture at 4 °C overnight to enrich RNA binding protein. The RNA that bound to FBL protein was collected by Proteinase K digestion, followed by purification and precipitation. For high-throughput 15 sequencing, input and RIP-ed RNA samples were shipped to BGI for library preparation and sequencing using the DNBseq platform. After mapping to the genome hg38 using tophat (version 2.1.1) with default parameter values and UCSC known genes as a reference, the software piranha (Uren et al., (2012). Bioinformatics 28, 3013-3020. 10.1093/bioinformatics/bts569) was used to analyze the RIP-seq data with default parameters. 20 The Homer annotatePeaks.pl script was used to annotate peaks from the RIP-seq data (Heinz et al., (2010). Mol Cell 38, 576-589.10.1016/j.molcel.2010.05.004). A P-value cutoff of 0.01 was used to define FBL-binding RNAs in the C4-2 cells. RNA immunoprecipitation (RIP)-qPCR 25 The RIP experiment was conducted using the EZ-Magna RIP kit (Millipore) following the User’s Guide. In brief, control or FBL-deficient C4-2 cells were lysed in RIP lysis buffer and incubated with anti-CPSF7 antibody and protein A/G magnetic beads mixture at 4 °C overnight to enrich the RNA-bound CPSF7 proteins. The CPSF7-associated RNAs were extracted by Proteinase K digestion, followed by purification and reverse transcription into cDNA. Then, the 30 qPCR assay was performed to measure the %Input of selected mRNA candidates in each group using the primers shown in Supplementary Table 6. 120 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 Functional enrichment analysis of gene groups KEGG functional enrichment analysis was performed using the R package clusterProfiler (v4.0.5) (Wu, et al. (2021). clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb) 2, 100141.10.1016/j.xinn.2021.100141). Gene Set Enrichment 5 Analysis was performed using the GSEA Java software (v4.0.3) (Subramanian et al., (2005). Proc Natl Acad Sci U S A 102, 15545-15550.10.1073/pnas.0506580102). Overlap analysis between two gene sets was performed using the R package GeneOverlap (Shen, L. (2014). GeneOverlap: An R package to test and visualize gene overlaps. R Package). 10 Analysis of sequence features and APA of 2’-O-Methylated transcripts. To analyze the mRNA features of 2’-O-Methylated transcripts, mRNA features were used including length of UTRs, CDS, and exon transcripts in a public dataset (Agarwal et al., Genome Biol 23, 245). The ARE score of 3′UTR was calculated by the software AREscore (Spasic et al., (2012). PLoS Genet 8, e1002433.10.1371/journal.pgen.1002433) and miRNA 15 binding sites of UTRs and CDS from the miRWalk database (Sticht et al., (2018). PLoS One 13, e0206239.10.1371/journal.pone.0206239) were downloaded. Dapars2 was used to perform APA analysis with the default parameters (Xia et al., (2014). Nat Commun 5, 5274. 10.1038/ncomms6274; Li, et al. (2021). Nat Genet 53, 994-1005.10.1038/s41588-021-00864-5). A gene was considered to be regulated by FBL if its PDUI values from both replicates were 20 lower or greater in FBL-knockdown cells than in the control cells and if the difference in average PDUI values was larger than 0.1. In silico screening of RNA binding proteins co-localized with Nm CLIP-seq database of RNA binding proteins in HEK293/HEK293T cells were retrieved 25 from the POSTAR3 database (Zhao et al., Nucleic Acids Res 50, D287-D294), and the overlapped regions of each file were merged using “bedtools merge”. These regions were overlapped with Nm sites in HEK293T cells. Fisher exact test was performed to determine the significance of the overlap between the binding regions of the RNA binding protein and the Nm sites. An odd ratio of more than 1.5 and an adjusted P-value of less than 0.01 were cutoffs to 30 define a significant overlap. Analyze the overlap of Nm sites with reported Nm sites and other RNA modification sites RNA modification sites, including the m6A (Hu, et al. (2022). Nat Biotechnol 40, 1210- 1219.10.1038/s41587-022-01243-z), A to I editing (Chung et al., (2018). Cell 172, 811-824 121 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 e814.10.1016/j.cell.2017.12.038), Ψ (Dai, et al. (2023). Nat Biotechnol 41, 344-354. 10.1038/s41587-022-01505-w), m5C (Sun et al., (2019). Epigenomics 11, 439-453. 10.2217/epi-2018-0169), m7G (Zhang et al., Mol Cell 74, 1304-1316 e1308), and Nm (Chen et al., Cell Res 33, 727-730) sites, from HEK293T cells with single base resolution were collected 5 from the associated literature. These sites in different human genome versions were lifted to the same genome version, hg38, using LiftOver in the UCSC genome browser: https://genome.ucsc.edu. Other Embodiments 10 From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed 15 elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. 20 122

Claims

ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 What is claimed is: 1. An in vivo method of stabilizing an mRNA, the method comprising contacting an mRNA with a heterologous 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA 5 is modified with a 2’-O-methylation, thereby producing a modified mRNA. 2. An in vitro method of stabilizing an mRNA, the method comprising contacting an mRNA with a 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA. 10 3. A method of stabilizing an mRNA, the method comprising contacting an mRNA with a 2’-O-methyltransferase polypeptide, such that an internal site of the mRNA is modified with a 2’-O-methylation, thereby producing a modified mRNA, wherein the 2’-O-methyltransferase polypeptide comprises a polypeptide selected from the group consisting of: cap 15 methyltransferase 1 (CMTR1), cap methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA 20 methyltransferase 11 homolog (TRMT11), tRNA methyltransferase 13 homolog (TRMT13), and tRNA methyltransferase 44 homolog (TRMT44). 4. The method of claim 3, wherein the method is performed in-vitro or in-vivo. 25 5. The method of claim 3 or 4, wherein the 2’-O-methyltransferase is heterologous. 6. The method of any one of claims 1-5, wherein the 2’-O-methyltransferase comprises a fibrillarin (FBL) polypeptide. 30 7. The method of any one of claims 1-5, wherein the 2’-O-methyltransferase comprises a FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3) polypeptide 8. The method of any one of claims 1-5, wherein the 2’-O-methyltransferase comprises a 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase (Spb1) polypeptide. 123 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 9. The method of claim 1 or 2, wherein the 2’-O-methyltransferase polypeptide comprises a polypeptide selected from the group consisting of: cap methyltransferase 1 (CMTR1), cap methyltransferase 2 (CMTR2), fibrillarin (FBL), fibrillarin like 1 (FBLL1), FtsJ RNA 2'-O- 5 methyltransferase 1 (FTSJ1), FtsJ RNA 2'-O-methyltransferase 3 (FTSJ3), HEN methyltransferase 1 (HENMT1), mitochondrial rRNA methyltransferase 1 (MRM1), mitochondrial rRNA methyltransferase 2 (MRM2 or FTSJ2), mitochondrial rRNA methyltransferase 3 (MRM3), tRNA guanosine 2 -O-methyltransferase (TARBP1), tRNA methyltransferase 11 homolog (TRMT11), tRNA methyltransferase 13 homolog (TRMT13), and 10 tRNA methyltransferase 44 homolog (TRMT44). 10. The method of any of claims 1-9, wherein the modified mRNA is modified in comparison to a reference mRNA, wherein the reference mRNA is an unmodified or wild type version of the modified mRNA. 15 11. The method of any one of claims 1-10, the method further comprising complexing the 2’- O-methyltransferase with a polynucleotide prior to contacting the mRNA with the 2’-O- methyltransferase, wherein the polynucleotide is at least partially complementary and/or capable of hybridizing to a target site within the mRNA. 20 12. The method of claim 11, wherein the polynucleotide is capable of directing the 2’-O- methyltransferase to the target site within the mRNA. 13. The method of claim 11 or 12, the method further comprising identifying one or more25 microRNA (miRNA) binding sites within the mRNA prior to complexing the 2’-O- methyltransferase with a polynucleotide, wherein the polynucleotide is at least partially complementary and/or capable of hybridizing to one or more of the identified miRNA binding sites. 30 14. A method of producing a stabilized mRNA, the method comprising using in-vitro transcription to produce a modified mRNA comprising one or more 2’-O-methyl modifications at an internal site of the modified mRNA, 124 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 15. The method of claim 14, wherein the in-vitro transcription is performed using an RNA polymerase. 16. The method of claim 15, wherein the RNA polymerase is a T7 RNA polymerase. 5 17. The method of claim 15 or 16, wherein the RNA polymerase comprises one or more thermostabilizing mutations. 18. The method of claim 17, wherein the thermostabilizing mutations comprise one or more 10 mutations selected from the group consisting of: R425X, R425C, G542X, G542V, Y639X, Y639G, Y639F, Y639V, H784X, H784A, H784G, H784S, E593G, V685A, I119V, G225S, K333N, D366N, F400L, S661G, F880Y, H772R, S430P, N433T, S633P, F849I, and P266L, wherein X is any amino acid. 15 19. The method of any of claims 14-18, wherein the modified mRNA is modified in comparison to a reference mRNA, wherein the reference mRNA is an unmodified or wild type version of the modified mRNA. 20. The method of any one of claims 1-19, wherein the modified mRNA is characterized as 20 having an increased half life of about 10% to about 300% as compared to a reference mRNA. 21. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of about 50% to about 300% as compared to a reference mRNA. 25 22. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of about 50% to about 250% as compared to a reference mRNA. 23. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of about 50% to about 200% as compared to a reference mRNA. 30 24. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of at least about 10%, at least about 20%, at least about 30%, or at least about 40% as compared to a reference mRNA. 125 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 25. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of at least about 50% as compared to a reference mRNA. 26. The method of any one of claims 1-19, wherein the modified mRNA is characterized as 5 having an increased half life of at least about 70% as compared to a reference mRNA. 27. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of at least about 90% as compared to a reference mRNA. 10 28. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of at least about 100% as compared to a reference mRNA. 29. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of at least about 150% as compared to a reference mRNA. 15 30. The method of any one of claims 1-19, wherein the modified mRNA is characterized as having an increased half life of at least about 200% as compared to a reference mRNA. 31. The method of any one of claims 1-19, wherein the modified mRNA is characterized as 20 having an increased half life of at least about 250% as compared to a reference mRNA. 32. An in vitro transcribed or chemically synthesized single stranded polynucleotide comprising one or more modified ribonucleotides comprising a 2’-O-methyl modification. 25 33. The single stranded polynucleotide of claim 32, wherein the polynucleotide is an RNA molecule or a DNA/RNA hybrid. 34. The single stranded polynucleotide of claim 32, wherein at least about 10% of the ribonucleotides comprise a 2’-O-methyl modification. 30 35. The single stranded polynucleotide of claim 32, wherein at least about 15% of the ribonucleotides comprise a 2’-O-methyl modification. 126 ATTORNEY DOCKET NO.167705-036001/PCT CLIENT REF. NO. CMCC 4423 DATE OF ELECTRONIC DEPOSIT: April 4, 2025 36. The single stranded polynucleotide of claim 32, wherein at least about 20% of the ribonucleotides comprise a 2’-O-methyl modification. 37. The single stranded polynucleotide of claim 32, wherein at least about 25% of the 5 ribonucleotides comprise a 2’-O-methyl modification. 38. An in vitro transcribed or chemically synthesized single stranded polynucleotide comprising one or more modified ribonucleotides comprising a 2’-O-methyl modification at a non-naturally occurring position. 10 127
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170130255A1 (en) * 2012-08-14 2017-05-11 Modernatx, Inc. Enzymes and polymerases for the synthesis of rna
US20230210960A1 (en) * 2020-06-18 2023-07-06 Korea University Research And Business Foundation New use of cmtr1 having sirna production and function enhancing activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170130255A1 (en) * 2012-08-14 2017-05-11 Modernatx, Inc. Enzymes and polymerases for the synthesis of rna
US20230210960A1 (en) * 2020-06-18 2023-07-06 Korea University Research And Business Foundation New use of cmtr1 having sirna production and function enhancing activity

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