WO2009006584A2 - Procédés et compositions pour identifier des produits de transcription d'arn cellulaire - Google Patents

Procédés et compositions pour identifier des produits de transcription d'arn cellulaire Download PDF

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WO2009006584A2
WO2009006584A2 PCT/US2008/069178 US2008069178W WO2009006584A2 WO 2009006584 A2 WO2009006584 A2 WO 2009006584A2 US 2008069178 W US2008069178 W US 2008069178W WO 2009006584 A2 WO2009006584 A2 WO 2009006584A2
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rna
sample
phosphatased
ligased
modified
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PCT/US2008/069178
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WO2009006584A3 (fr
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Linda F. Van Dyk
Kevin W. Diebel
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The Regents Of The University Of Colorado
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • MicroRNAs are small noncoding RNAs approximately 21-24 nucleotides in length that have the ability to post-transcriptionally down-regulate the expression of mRNAs that contain complementary target sequences within their 3' untranslated region (3'UTR) (Bartel, D. P. Cell 116:281-297(2004)). Close to 500 miRNAs have been predicted to be encoded within the human genome and each of these miRNAs is estimated to regulate the expression of 5-10 mRNAs (Sarnow, P. et al., Nat. Rev. Microbiol. 4:651-659 (2006)).
  • miRNAs Post-transcriptional gene regulation by miRNAs is a key biological process regulating a significant proportion of all gene expression (Cullen, B. R., Nat. Genet. 38 Suppl:S25-S30 (2006)). Although the exact functions of most individual miRNA genes remain unknown, work completed thus far has demonstrated that miRNA functions play key roles in embryonic development, hematopoiesis, stress resistance, fat metabolism, brain morphogenesis, as well as possible roles in cancer development (Gregory, R. I. et al.,
  • Biogenesis of mature miRNAs is typically described as beginning with the transcription of the primary-miRNA (pri-miRNA) transcript via RNA polymerase II (pol II) from either single or clustered transcriptional units resulting in long monocistronic, dicistronic, or polycistronic transcripts (Lee, Y, et al., EMBO J. 23:4051-4060 (2004); Cai, X. et al., RNA 10: 1957-1966 (2004)).
  • Stem-loop structures within the pri-miRNA transcript that are efficiently processed into mature miRNAs consist of stems ⁇ 33bp in length, a terminal loop, and flanking ssRNA segmanets (Han, J.
  • pre-miRNA an intermediately processed form of the miRNA known as the precursor-miRNA (pre-miRNA) which is exported from the nucleus to the cytoplasm by exportin-5 (Zeng, Y. et al., Nucleic Acids Res. 32:4776-4785 (2004); Yi, R. et al., Genes Dev. 17:3011-3016 (2003)).
  • pre-miRNA is cleaved by another RNaseIII enzyme family member, Dicer, to yield maturely processed RNA duplexes (Hutvagner, G. et al., Science 293:834-838 (2001)).
  • the strand of the RNA duplex containing the weakest thermodynamic stability at its 5' terminus becomes the 'guide strand', or mature miRNA, and is selectively loaded into the miRNA effector complex, known as the miRNA-containing RNA-induced silencing complex (miRISC) (Maniataki, E. et al., Genes Dev. 19:2979-2990 (2005); Khvorova, A. et al., Cell 115:209-216 (2003)). Interactions between the miRISC and target mRNAs lead to translational inhibition or degradation of the target mRNAs by mechanisms which are currently not fully understood.
  • miRISC miRNA-containing RNA-induced silencing complex
  • these gammaherpesviruses are large double-stranded DNA, lymphotropic viruses whose viral life-cycles are characterized by an initial acute/lytic phase of infection followed by the establishment of a life-long latent infection interlaced with short, controlled, bursts of lytic reactivation from latency within healthy hosts.
  • ⁇ HV68 a natural pathogen of wild murid rodents serves as a convenient small animal model used in the investigation of gammaherpesvirus pathogenesis that allows for the complete characterization of the full range of gammaherpesviral infection, from acute infection to the maintenance of latency, reactivation, and chronic disease
  • Ehtisham S. et al., J. Virol. 67:5247-5252 (1993); Tibbetts, S. A. et al., J. Virol. 76:7125-7132 (2002); Speck, S. H. et al., Curr.
  • miRNAs within the ⁇ HV68 genome were originally discovered by cloning and sequencing gel-purified small RNA from a total RNA isolation of a ⁇ HV68 latently infected mouse B cell lymphoma cell line (Pfeffer, S. et al., Nat. Methods 2:269-276 (2005)).
  • vtR ⁇ A transcripts have previously been shown to 1 ) contain the canonical R ⁇ A polymerase III (pol III), type 2 promoter elements required for transcription initiation by R ⁇ A pol III, 2) lack any upstream TATA boxes, 3) contain up to 75% sequence homology to known tR ⁇ As genes previously identified within mammalian genomes, and 4) are transcribed during all phases of the viral lif-cycle, both latent and lytic (Bowden, R. J. et al., J. Gen. Virol. 78 ( Pt 7):1675-1687 (1997); Ebrahimi, B. et al., J. Gen. Virol. 84:99-109 (2003); Simas, J.
  • miRNAs produced by RNA pol III transcription of AIu elements in mammalian genomes and the adenovirus VAI gene (Borchert, G. M. et al., Nat. Struct. MoI. Biol. (2006); Sano, M. et al., FEBS Lett. 580: 1553-1564 (2006)). Also, modes of post-transcriptional processing to generate both tRNAs and mature miRNAs from a single transcript by RNA processing pathways, currently thought to be independent of one another, has never before been defined.
  • the present invention provides a method of determining whether a population of cellular RNAs comprises 5'-monophosphate modified cellular RNA, 5'-OH cellular RNA, 5'-triphosphate modified cellular RNA, or 5'-CAP modified cellular RNA.
  • the invention provides method of determining whether a population of cellular RNAs comprises 5'-monophosphate modified 3'-OH cellular RNA, 5'- OH 3'-OH cellular RNA, 5 '-triphosphate modified 3'-OH cellular RNA, or 5'-CAP modified 3'-OH cellular RNA.
  • an isolated ribonucleic acid with the following sequence is provided: 5'-CUA CGA CAG UCA CCA CCG AUA CCC UGU ACU ACG CAC CAC GAU GAA A-3'.
  • the present invention provides a kit for determining whether a population of cellular RNAs include 5'-monophosphate modified cellular RNA, 5'-OH cellular RNA, 5'-triphosphate modified cellular RNA, or 5'-CAP modified cellular RNA.
  • the kit includes a ligase, a phosphatase, a pyrophosphatase, and a 5'-OH RNA linker.
  • the present invention provides a kit for determining whether a population of cellular RNAs include 5'-monophosphate modified 3'-OH cellular RNA, 5'- OH 3'-OH cellular RNA, 5'-triphosphate modified 3'-OH cellular RNA, or 5'-CAP modified 3'-OH cellular RNA.
  • the kit includes a ligase, a phosphatase, and a pyrophosphatase.
  • Figure 1 illustrates the ⁇ HV68 pol III coding locus.
  • A. The left end of the ⁇ HV68 genome.
  • B. RNA pol III type 2 promoter elements for each ⁇ HV68 pol III gene.
  • C. 2D graphical representation of the ⁇ HV68 pol III genes 1, 4, and 5.
  • D. Predicted RNA folds of the theoretical ⁇ HV68 pol III transcripts.
  • FIG. 2 illustrates the detection of processed viral miRNAs throughout multiple states of infection using reverse ligation mediated-reverse transcription-polymerase chain reaction (RLM-RT-PCR).
  • RLM-RT-PCR reverse ligation mediated-reverse transcription-polymerase chain reaction
  • Figure 3 illustrates the ⁇ -amanitin verification of RNA pol III transcription from the ⁇ HV68 genome.
  • A. RT-PCR analysis of ⁇ HV68 pol III- 1 , 4, and 5 in ⁇ -amanitin treated 293T cells.
  • B. RT-PCR analysis of the minimal ⁇ HV68 pol III-l transcript that can contain the miR-Ml-1 sequences in ⁇ -amanitin treated 293T cells.
  • FIG 4 illustrates RLM-RT-PCR amplification of RNA molecules with various 5' end biochemical modifications.
  • A. The 5'HMCT Assay. Total RNA contains many 5' end biochemical groups including: monophosphates, caps, and triphosphates.
  • B. Cellular controls ⁇ -actin and tRNA ⁇ RLM-RT-PCR products within the 5 1 MCT assay.
  • Figure 5 illustrates the RARE Assay. 1. Total RNA can either be left untreated to identify RNA molecules with 5' monophosphate groups, or subjected to the 5'MCT assay as previously described. 2.
  • RNA molecules undergo a reverse ligation reaction via T4 RNA Ligase in the absence of a synthetic linker oligo to circularize the RNA. 3.
  • the circular RNA is RT-PCR amplified with inverse primer sets to yield a PCR product that reveals the joined 5' and 3' ends of individual RNA molecules after cloning and sequencing of the PCR product.
  • Figure 6 illustrates the mapping and structure prediction of the ⁇ HV68 pol III transcripts.
  • A. 3T3 cells were infected with 5 PFU/cell of ⁇ HV68 and total RNA was harvested at 24 hours post-infection and subjected to the 5'MCT / RARE assay combination.
  • B. ⁇ HV68 pol III-l predicted secondary structures.
  • C. ⁇ HV68 pol III-4 predicted secondary structure.
  • D. ⁇ HV68 pol III-5 predicted secondary structures.
  • Figure 7 illustrates the similarity of the adenovirus VAl and ⁇ HV68 pol III-l predicted secondary structures.
  • Figure 8 is a table illustrating the 5'MCT / RARE Assay mapping of the ⁇ HV68 pol III transcripts.
  • Figure 9 A. and B. list oligonucleotides in the order presented within the figures of this application.
  • Figure 10 illustrates a polyacrylamide gel stained with ethidium bromide showing ligation of differing ratios of 5'-OH RNA linker to 5 '-monophosphate oligonucleotides.
  • Figure 11 illustrates a polyacrylamide gel stained with ethidium bromide showing ligation of differing amounts of 5'-OH RNA linker in 5 ⁇ g of total RNA isolated from 3T3 mouse cells.
  • Figure 12 illustrates a polyacrylamide gel stained with ethidium bromide showing ligation of 5'-OH RNA linker with various 5' end modifications.
  • Figure 13 illustrates a polyacrylamide gel stained with ethidium bromide showing ligation of 5'-OH RNA linker with kinase treated cellular RNA containing various 5' end modifications.
  • Figure 14 illustrates a polyacrylamide gel stained with ethidium bromide showing ligation of 5'-OH RNA linker with phosphatase and pyrophosphatase treated cellular RNA containing various 5' end modifications.
  • Figure 15 illustrates a polyacrylamide gel stained with ethidium bromide showing ligation of 5'-OH RNA linker with phosphatase treated RNA containing various 5' end modifications.
  • cellular RNA means RNA (ribonucleic acid) found in prokaryotic and/or eukaryotic cells, either isolated or as part of an multicellular construct (e.g. organism or organ), including viral RNAs that become part of a cellular RNA population.
  • a 5'-monophosphate modified RNA is an RNA having a monophosphate group at its 5' end (i.e. covalently attached to the RNA 5'-OH group), whereas a 5'-triphosphate modified RNA refers to an RNA that having a triphosphate group at its 5' end.
  • 5'-CAP modified RNA is an RNA having a CAP group at its 5' end.
  • the 5' CAP is a modified guanine nucleotide added to the 5' end of the pre-mRNA using a 5'-triphosphate linkage as is well known in the art.
  • 5'-monophosphate modified 3'-OH RNA is a 5'- monophosphate modified RNA having a hydroxyl at its 3' end.
  • 5'-triphosphate modified 3'-OH RNA and 5'-CAP modified 3'-OH RNA are 5'-triphosphate modified RNA and 5'-CAP modified RNA with 3' hydroxyls, respectively.
  • a "population of cellular RNAs" is a plurality of RNAs that are obtained from cells.
  • miRNA precursor As used herein, the term "miRNA precursor,” “microRNA precursor,” or “precursor thereof in reference to a particular microRNA (i.e. miRNA) refers broadly to any precursor which through processing in a cell results in the specified miRNA. The term thus includes the corresponding pri-miRNA, pre-miRNA or variant thereof. In some embodiments, the precursor is the corresponding pri-miRNA or pre-miRNA.
  • the pre- miRNA sequence may include, for example, from 45-90, 60-80 or 60-70 nucleotides.
  • the sequence of the pre-miRNA may include the entire miRNA sequence, or be that of a pri- miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA.
  • the sequence of the pre-miRNA may comprise the sequence of a hairpin loop.
  • the pri-miRNA sequence may comprise from 45-250, 55-200, 70-150 or 80-100 nucleotides.
  • the sequence of the pri-miRNA may include the pre-miRNA or miRNA.
  • the pri-miRNA may also include a hairpin structure (e.g. from 37-50 nucleotides).
  • a "5'-OH RNA linker” is a single stranded RNA having an unmodified 5' and 3' ends (i.e. 5'-OH and 3'-OH).
  • the 5'-OH RNA linker may be modified to include chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid.
  • Such modifications include, but are not limited to, phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping moieties.
  • phosphodiester group modifications e.g., phosphorothioates, methylphosphonates
  • 2'-position sugar modifications e.g., 2-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base
  • a 2'deoxy nucleic acid linker is a divalent nucleic acid of any appropriate length and/or internucleotide linkage wherein the nucleotides are 2'deoxy nucleotides.
  • "Complementary,” as used herein, refers to the capacity for precise pairing of two nucleobases (e.g. A to T (or U), and G to C) regardless of where in the nucleic acid or miRNA or miRNA precursor the two are located.
  • nucleic acid and miRNA or miRNA precursor are “substantially complementary” to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases that can hydrogen bond with each other.
  • substantially complementary is used to indicate a sufficient degree of precise pairing over a sufficient number of nucleobases such that stable and specific binding occurs between the nucleic acid and an miRNA or miRNA precursor.
  • substantially complementary thus means that there may be one or more mismatches between the nucleic acid and the miRNA or miRNA precursor when they are aligned, provided that stable and specific binding occurs.
  • mismatch refers to a site at which a nucleobases in the nucleic acid and a nucleobases in the miRNA or precursor with which it is aligned are not complementary.
  • the nucleic acid and miRNA or miRNA precursor are "perfectly complementary" to each other when the nucleic acid is fully complementary to the miRNA or miRNA precursor across the entire length of the nucleic acid.
  • RNA transcripts derived from any cell are novel and powerful methods for identification of 5' and 3' modifications of RNA transcripts derived from any cell.
  • One skilled in the art will immediately recognize the broad applicability and utility of these methods, for example in developing RNA transcript maps.
  • the utility of these methods have been specifically exemplified in reference to RNA pol III transcripts as set forth below in the Examples (section IV).
  • the present invention provides a method (also referred to herein as the 5'HMCT method) of determining whether a population of cellular RNAs includes 5'- monophosphate modified cellular RNA, 5'-OH cellular RNA, 5'-triphosphate modified cellular RNA, or 5'-CAP modified cellular RNA.
  • the method includes contacting a first sample of the population with a first 5'-OH RNA linker and a ligase thereby forming a ligased first sample (i.e. a sample that has been contacted with a ligase).
  • the ligased first sample is subjected to 5'-OH RNA amplification thereby forming a ligased amplified first sample (i.e. a ligased first sample subjected to 5'-OH RNA amplification).
  • the ligased amplified first sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the ligased amplified first sample thereby determining whether 5'-monophosphate modified cellular RNA is present in the population.
  • the method further includes contacting a second sample of the population with a kinase and a nucleotide triphosphate thereby forming a kinased second sample (i.e. a sample that has been contacted with a kinase).
  • the kinased second sample is contacted with a second 5'-OH RNA linker and a ligase thereby forming a kinased ligased second sample (i.e. a kinased sample that has been contacted with a ligase).
  • the kinased ligased second sample is subjected to 5'-OH RNA amplification thereby forming a kinased ligased amplified second sample (i.e.
  • a kinased ligased second sample subjected to 5'-OH RNA amplification.
  • the kinased ligased amplified second sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the kinased ligased amplified second sample thereby determining whether 5'-OH cellular RNA is present in the population.
  • the method further includes contacting a third sample of the population with a phosphatase thereby forming a phosphatased third sample (i.e. a sample that has been contacted with a phosphatase). At least a first portion of the phosphatased third sample is contacted with a kinase and a nucleotide triphosphate thereby forming a phosphatased kinased third sample (i.e. a phosphatased sample that has been contacted with a kinase).
  • the phosphatased kinased third sample is contacted with a third 5'-OH RNA linker and a ligase thereby forming a phosphatased kinased ligased third sample (i.e. a phosphatased kinased ample that has been contacted with a ligase).
  • the phosphatased kinased ligased third sample is subjected to 5'-OH RNA amplification thereby forming a phosphatased kinased ligased amplified third sample (i.e. a phosphatased kinased ligased third sample subjected to 5'-OH RNA amplification).
  • the phosphatased kinased ligased amplified third sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the phosphatased kinased ligased amplified third sample thereby determining whether 5'-triphosphate modified cellular RNA is present in the population.
  • the method further includes contacting a second portion of the phosphatased third sample with a pyrophosphatase thereby forming a phosphatased pyrophosphatased third sample (i.e. a phosphatased sample contacted with a pyrophosphatase).
  • the phosphatased pyrophosphatased third sample is contacted with a fourth 5'-OH RNA linker and a ligase thereby forming a phosphatased pyrophosphatased ligased third sample (i.e. a phosphatased pyrophosphatased sample contacted with a ligase).
  • the phosphatased pyrophosphatased ligased third sample is subjected to 5'-OH RNA amplification thereby forming a phosphatased pyrophosphatased ligased amplified third sample (i.e. a phosphatased pyrophosphatased ligased third sample subjected to 5'-OH RNA amplification).
  • the phosphatased pyrophosphatased ligased amplified third sample is analyzed to determine whether 5'-OH RNA amplification products are present in the phosphatased pyrophosphatased ligased amplified third sample thereby determining whether 5'-CAP modified cellular RNA is present in the population.
  • the method further includes contacting a fourth sample with a phosphatase thereby forming a phosphatased fourth sample.
  • the phosphatased fourth sample is contacted with a pyrophosphatase thereby forming a phosphatased pyrophosphatased fourth sample.
  • the phosphatased pyrophosphatased fourth sample is contacted with a fourth 5'-OH RNA linker and a ligase thereby forming a phosphatased pyrophosphatased ligased fourth sample.
  • the phosphatased pyrophosphatased ligased fourth sample is subjected to 5'-OH RNA amplification thereby forming a phosphatased pyrophosphatased ligased amplified fourth sample.
  • the phosphatased pyrophosphatased ligased amplified fourth sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the phosphatased pyrophosphatased ligased amplified fourth sample thereby determining whether 5'-CAP modified cellular RNA is present in the population.
  • the first 5'-OH RNA linker, second 5'-OH RNA linker, third 5'-OH RNA linker, and/or fourth 5'-OH RNA linker are independently the same or different.
  • each of the linkers contain different nucleotide sequence corresponding to a particular 5' RNA characteristic (i.e. 5'-OH, 5'monophoshate, 5'-triphosphate or 5'-CAP). Where the respective linkers contain different sequences, the determination of whether 5'- OH RNA amplification products are present in a particular sample may be simplified.
  • the methods of the present invention include sequencing the first 5'-OH RNA linker, second 5'-OH RNA linker, third 5'-OH RNA linker, and/or fourth 5'-OH RNA linker.
  • the 5 '-monophosphate modified cellular RNA, the 5'- triphosphate modified cellular RNA, and the 5'-CAP modified cellular RNA are microRNA.
  • a population of cellular RNAs comprises 5'-monophosphate modified 3'-OH cellular RNA, 5'-OH 3'-OH cellular RNA, 5'-triphosphate modified 3'-OH cellular RNA, or 5'-CAP modified 3'-OH cellular RNA (also referred to herein as the RARE assay).
  • the method includes contacting a first sample of the population with a ligase thereby forming a ligased first sample. Where the sample contains 5'-monophosphate modified 3'-OH cellular RNA, circularized RNA is formed.
  • the ligased first sample is subjected to circularized RNA amplification thereby forming a ligased amplified first sample.
  • the ligased amplified first sample is analyzed to determine whether circularized RNA amplification products are present in the ligased amplified first sample thereby determining whether 5'-monophosphate modified 3'-OH cellular RNA is present in the population.
  • the method also includes contacting a second sample of the population with a kinase and a nucleotide triphosphate thereby forming a kinased second sample.
  • the kinased second sample is contacted with a ligase thereby forming a kinased ligased second sample.
  • the sample contains 5'-OH 3'-OH cellular RNA
  • circularized RNA is formed.
  • the kinased ligased second sample is subjected to circularized RNA amplification thereby forming a kinased ligased amplified second sample.
  • the kinased ligased amplified second sample is analyzed to determine whether circularized RNA amplification products are present in the kinased ligased amplified second sample thereby determining whether 5'-OH 3'-OH cellular RNA is present in the population.
  • the method further includes contacting a third sample of the population with a phosphatase thereby forming a phosphatased third sample. At least a portion of the phosphatased third sample is contacted with a kinase and a nucleotide triphosphate thereby forming a phosphatased kinased third sample. The phosphatased kinased third sample is contacted with a ligase thereby forming a phosphatased kinased ligased third sample. Where the sample contains 5'-triphosphate modified 3'-OH cellular RNA, circularized RNA is formed.
  • the phosphatased kinased ligased third sample is subjected to circularized RNA amplification thereby forming a phosphatased kinased ligased amplified third sample.
  • the phosphatased kinased ligased amplified third sample is analyzed to determine whether circularized RNA amplification products are present in the phosphatased kinased ligased amplified third sample thereby determining whether 5'-triphosphate modified 3'-OH cellular RNA is present in the population.
  • the method further includes contacting a second portion of the phosphatased third sample with a pyrophosphatase thereby forming a phosphatased pyrophosphatased third sample.
  • the phosphatased pyrophosphatased third sample is contacted with a ligase thereby forming a phosphatased pyrophosphatased ligased third sample.
  • the sample contains 5'-CAP modified cellular RNA, circularized RNA is formed.
  • the phosphatased pyrophosphatased ligased third sample is subjected to circularized RNA amplification thereby forming a phosphatased pyrophosphatased ligased amplified third sample.
  • the phosphatased pyrophosphatased ligased amplified third sample is then analyzed to determine whether circularized RNA amplification products are present in the phosphatased pyrophosphatased ligased amplified third sample thereby determining whether 5'-CAP modified cellular RNA is present in the population.
  • the method further includes contacting a fourth sample with a phosphatase thereby forming a phosphatased fourth sample.
  • the phosphatased fourth sample is contacted with a pyrophosphatase thereby forming a phosphatased pyrophosphatased fourth sample.
  • the phosphatased pyrophosphatased fourth sample is contacted with a fourth 5'-OH RNA linker and a ligase thereby forming a phosphatased pyrophosphatased ligased fourth sample.
  • the sample contains 5'-CAP modified cellular RNA
  • circularized RNA is formed.
  • the phosphatased pyrophosphatased ligased third sample is subjected to circularized RNA amplification thereby forming a phosphatased pyrophosphatased ligased amplified fourth sample.
  • the phosphatased pyrophosphatased ligased amplified fourth sample is then analyzed to determine whether circularized RNA amplification products are present in the phosphatased pyrophosphatased ligased amplified fourth sample thereby determining whether 5'-CAP modified cellular RNA is present in the population.
  • the 5'-monophosphate modified 3'-OH cellular RNA, the 5'-triphosphate modified 3'-OH cellular RNA, 5'-OH 3'-OH cellular RNA, and/or the 5'- CAP modified 3'-OH cellular RNA are precursors of microRNA or non-tRNA polymerase III transcripts.
  • the nucleotide triphosphate is adenosine triphosphate (ATP).
  • the population of cellular RNA is derived from a cell infected with a virus.
  • the virus may be a herpesvirus.
  • the cells are from a single source (e.g. a single organism, organ, or multicellular construct, or a group of the same or different cell types exposed to the same set of environmental conditions). The cells may also be from different sources.
  • RNA is extracted from the cell using well established RNA extraction techniques to form a population of cellular RNA.
  • the ligase is T4 RNA ligase
  • the phosphatase is Antarctic phosphatase
  • the kinase is T4 polynucleotide kinase
  • the pyrophosphatase is tobacco acid pyrophosphatase.
  • the phosphatase may also be calf intestinal phosphatase (CIP), shrimp alkaline phosphatase (SAP), or APEX phosphatase.
  • analyzing the amplified sample to determine whether 5'-OH RNA amplification products are present would further involve comparison to and exclusion of the identified 5'-monophosphate modified cellular RNA.
  • these comparisons may be simply by sequencing and quantitating the respective linkers corresponding to specific 5'-end modifications (including 5'-OH cellular RNA).
  • 5'-OH phosphatased cellular RNA By contacting a sample containing 5'-mono- and triphosphate cellular RNA with a phosphatase, 5'-OH phosphatased cellular RNA is generated.
  • the cellular RNAs having a 5'-CAP moiety are unaffected by the phosphatase.
  • the 5'-OH phosphatased cellular RNA is treated with a kinase and a nucleotide triphosphate to form 5'- monophosphate phosphatased cellular RNA.
  • the 5'-monophosphate phosphatased cellular RNA is ligated to a 5'-OH RNA linker with a ligase to form a 5'-OH phosphatased ligated RNA.
  • Amplification and detection of this 5'-OH phosphatased ligated RNA identifies the 5'-triphosphate modified cellular RNA. Because the phosphatase will cleave any 5'-mono- and triphosphates from cellular RNAs, identification of the 5'-triphosphate modified cellular RNA (i.e. analyzing the amplified sample to determine whether 5'-OH RNA amplification products are present) may involve comparison to and exclusion of the identified 5'- monophosphate modified cellular RNA. Moreover, where 5'-OH cellular RNA is present in the population of cellular RNAs, identification of the 5'-triphosphate modified cellular RNA would further involve comparison to and exclusion of the identified 5'-OH cellular RNA.
  • the method may be modified in which the method includes determining whether a population of cellular RNAs comprises 5'-monophosphate modified cellular RNA, 5'-triphosphate modified cellular RNA, or 5'- CAP modified cellular RNA. This method may also be referred to herein as the 5'MCT method.
  • the method includes contacting a first sample of the population with a first 5'-OH RNA linker and a ligase thereby forming a ligased first sample.
  • the ligased first sample is subjected to 5'-OH RNA amplification thereby forming a ligased amplified first sample.
  • the ligased amplified first sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the ligased amplified first sample thereby determining whether 5'-monophosphate modified cellular RNA is present in the population.
  • the method further comprises contacting a second sample of the population with a phosphatase thereby forming a phosphatased second sample. At least a first portion of the phosphatased second sample is contacted with a kinase and a nucleotide triphosphate thereby forming a phosphatased kinased second sample. The phosphatased kinased second sample is contacted with a second 5'-OH RNA linker and a ligase thereby forming a phosphatased kinased ligased second sample.
  • the phosphatased kinased ligased second sample is subjected to 5'-OH RNA amplification thereby forming a phosphatased kinased ligased amplified second sample.
  • the phosphatased kinased ligased amplified second sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the phosphatased kinased ligased amplified second sample thereby determining whether 5'-triphosphate modified cellular RNA is present in the population.
  • the method further includes contacting a second portion of the phosphatased second sample with a pyrophosphatase thereby forming a phosphatased pyrophosphatased second sample.
  • the phosphatased pyrophosphatased second sample is contacted with a third 5'-OH RNA linker and a ligase thereby forming a phosphatased pyrophosphatased ligased second sample.
  • the phosphatased pyrophosphatased ligased second sample is subjected to 5'-OH RNA amplification thereby forming a phosphatased pyrophosphatased ligased amplified second sample.
  • the phosphatased pyrophosphatased ligased amplified second sample is analyzed to determine whether 5'-OH RNA amplification products are present in the phosphatased pyrophosphatased ligased amplified second sample thereby determining whether 5'-CAP modified cellular RNA is present in the population.
  • the method further includes contacting a third sample with a phosphatase thereby forming a phosphatased third sample.
  • the phosphatased third sample is contacted with a pyrophosphatase thereby forming a phosphatased pyrophosphatased third sample.
  • the phosphatased pyrophosphatased third sample is contacted with a third 5'- OH RNA linker and a ligase thereby forming a phosphatased pyrophosphatased ligased third sample.
  • the phosphatased pyrophosphatased ligased third sample is subjected to 5'- OH RNA amplification thereby forming a phosphatased pyrophosphatased ligased amplified third sample.
  • the phosphatased pyrophosphatased ligased amplified third sample is then analyzed to determine whether 5'-OH RNA amplification products are present in the phosphatased pyrophosphatased ligased amplified third sample thereby determining whether 5'-CAP modified cellular RNA is present in the population.
  • the method described in the immediately preceding paragraphs is equally applicable to RARE assay methods described above.
  • the first 5'-OH RNA linker, second 5'-OH RNA linker, and/or third 5'-OH RNA linker are independently the same or different.
  • a method is provided to identify non-tRNA polymerase III transcripts in a population of cellular RNAs.
  • the method includes treatment of a first portion of cellular RNAs with a ligase to form a circularized RNA. Amplification of the circularized (e.g. with a RNA polymerase III promoter primer) and detection of this circularized RNA identifies a first non-tRNA polymerase III transcript originally containing a 5'-monophosphate. A second portion of the population of cellular RNAs is treated with a phosphatase to form a 5'-OH phosphatased cellular RNA.
  • the 5'-OH phosphatased cellular RNA is treated with a kinase and nucleotide triphosphate to form a 5'-monophosphate phosphatased cellular RNA.
  • the 5'-monophosphate phosphatased cellular RNA is treated with a ligase to form a circularized phosphatased RNA.
  • Amplification of the circularized phosphatased RNA e.g. with a RNA polymerase III promoter primer
  • detection of the circularized phosphatased RNA identifies a second non-tRNA polymerase III transcript originally containing a 5'-triphosphate.
  • 5'-OH RNA amplification refers to any method capable of increasing the number of RNAs transcripts (i.e. 5'- OH RNA amplification products) derived from 5'-OH RNA in a sample to detectable levels.
  • circularized RNA amplification refers to any method capable of increasing the number of RNAs (i.e. circularized RNA amplification products) derived from circularized RNAs (i.e. circular RNA) present in the sample to detectable levels.
  • RNA amplification method or circularized RNA amplification is reverse transcription-polymerase chain reaction (RT-PCR) amplification (e.g. RLM-RT-PCR).
  • RT-PCR reverse transcription-polymerase chain reaction
  • RLM-RT-PCR reverse transcription-polymerase chain reaction
  • a portion of a sample is needed for 5'-OH RNA amplification or circularized RNA amplification.
  • the 5'-OH RNA amplification products or circularized RNA amplification products may be detected using any applicable technique, including sequencing (e.g. as full or partial sequencing of the amplified RNAs), restriction digest analysis, or nucleic acid hybridization.
  • the present invention provides a 5'-OH RNA linker.
  • the term 5'-OH RNA linker refers to a RNA molecule of sufficient length to, upon ligation with a target RNA, from a product RNA molecule capable of being amplified using known techniques.
  • the sequence of the 5'-OH RNA linker is designed to avoid cross reactions and hybridization with members of the population of cellular RNAs.
  • the 5'-OH RNA is at least one nucleotide in length.
  • the 5'-OH RNA linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.
  • the 5'-OH RNA linker may also be an RNA from 5 to 100, 10 to 80, 20 to 70, 30 to 60, or about 50 nucleotides in length.
  • the ratio of the 5'-OH RNA linker to the 5'monophosphate RNA compound to which it is ligated is at least about 1 :2. In other embodiments, the ratio is about 1 :2, about 1 :3, or about 1 :4.
  • the 5'-OH RNA linker is an isolated RNA with the following sequence: 5'-CUA CGA CAG UCA CCA CCG AUA CCC UGU ACU ACG CAC CAC GAU GAA A-3'.
  • a 5'-OH Linker RNA is provided with a sequence perfectly complimentary to 5'-CUA CGA CAG UCA CCA CCG AUA CCC UGU ACU ACG CAC CAC GAU GAA A-3'.
  • the present invention provides a kit for determining whether a population of cellular RNAs include 5'-monophosphate modified cellular RNA, 5'-OH cellular RNA, 5'-triphosphate modified cellular RNA, and/or 5'-CAP modified cellular RNA.
  • the kit includes a ligase, a phosphatase, a pyrophosphatase, and a 5'-OH RNA linker.
  • the kit includes a control 5'-monophosphate modified cellular RNA, a control 5'-OH cellular RNA, a control 5'-triphosphate modified cellular RNA, and/or a control 5'-CAP modified cellular RNA.
  • the present invention provides a kit for determining whether a population of cellular RNAs include 5 '-monophosphate modified 3'-OH cellular RNA, 5'- OH 3'-OH cellular RNA, 5 '-triphosphate modified 3'-OH cellular RNA, and/or 5'-CAP modified 3'-OH cellular RNA.
  • the kit includes a ligase, a phosphatase, and a pyrophosphatase.
  • the kit includes a control 5'-monophosphate modified 3'-OH cellular RNA, a control 5'-OH 3'-OH cellular RNA, a control 5'- triphosphate modified 3'-OH cellular RNA, and/or a control 5'-CAP modified 3'-OH cellular RNA.
  • kits of the present invention may further include an RNase inhibitor (i.e. an agent such as a protein that decreases the enzymatic activity of one or more RNase enzymes).
  • an RNase inhibitor i.e. an agent such as a protein that decreases the enzymatic activity of one or more RNase enzymes.
  • the ligase is T4 RNA ligase
  • the phosphatase is Antarctic phosphatase
  • the kinase is T4 polynucleotide kinase
  • the pyrophosphatase is tobacco acid pyrophosphatase.
  • kits described herein may further include instructions setting forth the steps, properties and/or components of the methods described herein.
  • RLM-RT-PCR reverse ligation mediated-reverse transcription-polymerase chain reaction
  • Murine gammaherpesvirus 68 ( ⁇ HV68), a virus of wild murid rodents, encodes nine distinct miRNAs. Interestingly, these viral miRNA genes are all located immediately downstream of RNA polymerase III (pol III) type 2, A box and B box promoter elements, ⁇ -amanitin treatment of ⁇ HV68 infected cells confirms that these viral pri-miRNA transcripts are produced via RNA pol III transcription.
  • pol III RNA polymerase III
  • RLM-RT-PCR reverse ligation mediated-reverse transcription-polymerase chain reaction
  • VAI is a multifunctional viral transcript known to be processed into mature miRNAs in addition to disrupting a variety of key cellular pathways including cellular miRNA biogenesis and aspects of innate immunity.
  • the discovery of similarities in the biogenesis and structures between the ⁇ HV68 pol III transcripts and the adenovirus VAI gene has potentially uncovered an important class of viral genes that could have conserved biological functions across diverse viral families.
  • RNA pol III can transcribe human miRNAs (Borchert, G. M. et al., Nat. Struct. MoI. Biol. (2006)). Within this work the authors speculate that over 20% of all human miRNAs could be transcribed by RNA pol III, mainly from AIu repeat regions of the genome. AIu repeat transcripts contain the same general RNA pol III Type 2 promoter as the ⁇ HV68 pol III transcripts.
  • the methods of the present invention allow examination of the mammalian genome to identify pol III promoters upstream of other experimentally confirmed miRNAs and consideration of whether many more miRNAs are transcribed by RNA pol III than previously assumed.
  • ⁇ HV68 pol III transcripts do not encode tRNA precursors.
  • many computer predicted mammalian tRNA transcripts have not been experimentally defined as actual tRNA transcripts. Therefore, the methods provided herein allow for consideration as to whether computer predicted tRNA transcripts may actually be pol III transcripts, like those described here for the ⁇ HV68 pol III transcripts, of different and perhaps pleiotropic functions including being pri-miRNA transcripts.
  • ⁇ HV68 transcripts formally known as the vtRNA transcripts actually do not make RNAs that physically resemble tRNA structures.
  • ⁇ HV68 pol III transcripts Based on these newly mapped ⁇ HV68 pol III transcripts, no evidence was found for the presence of a tRNA-like molecule originating from the ⁇ HV68 genome. But a better than previously predicted substrate was found for miRNA processing by the miRNA processing machinery.
  • the data also indicated that ⁇ HV68 transcription of its RNA pol III genes involves a previously uncharacterized RNA pol III, type 2, promoter consisting of a series of overlapping A box promoter elements followed by a single B box promoter element. During ⁇ HV68 infection, these promoters originally identified due to their homology to tRNA pol III promoters, give rise to pri- miRNA transcripts shorter than originally predicted.
  • the methods of the present invention made possible the complete mapping of ⁇ HV68 pol III transcripts, and thereby revealed that the ⁇ HV68 transcript pol III-l has a remarkably similar secondary structure as compared with the VAI gene ( Figure 7.).
  • both of these viral genes are transcribed from RNA pol III type 2 promoters and encode miRNAs.
  • the adenovirus VAI gene is a 160 nucleotide long pol III transcript that is highly transcribed and can accumulate up to 10 8 copies within an infected cell (Mathews, M. B. et al, J. Virol. 65:5657-5662 (1991)).
  • VAI is known to bind to PKR and inhibit its normal activation by viral double-stranded RNAs, bypassing an important innate cellular immune response to viral infection.
  • PKR activation leads to global cellular down-regulation of translation through the phosphorylation of eukaryotic translation-initiation factor 2 ⁇ (eIF2 ⁇ ) (Mathews, M. B. et al., J. Virol. 65:5657- 5662 (1991)).
  • eIF2 ⁇ eukaryotic translation-initiation factor 2 ⁇
  • VAI also shares use of the nuclear-export receptor exportin-5 with cellular pre-miRNAs and appears to overwhelm the export machinery during infection, leading to a global down-regulation of cellular mature miRNAs (Yi, R. et al., Genes Dev. 17:3011-3016 (2003)).
  • VAI can suppress the activity of the cytoplasmic RNaseIII enzyme, Dicer, by functioning as a competitive substrate, further blocking the ability of the infected cell to generate endogenous mature miRNA products (Andersson, M. G. et al., /. Virol. 79:9556-9565 (2005); Lu, S. et al., J. Virol. 78:12868-12876 (2004)).
  • Dicer cytoplasmic RNaseIII enzyme
  • Being a substrate for Dicer results in cleavage of the VAI gene product and production of small VA RNAs (svaRNAs), which have been shown to be associated with RISC complexes during infection (Andersson, M. G. et al., J. Virol. 79:9556-9565 (2005)).
  • this single gene product is able to disrupt key events in innate cellular immunity and miRNA maturation as well as to generate miRNAs of its own.
  • no targets for specific down-regulation of cellular or viral genes have been shown to be associated with the svaRNA charged RISC complexes, it is conceivable that this could also be a function of the VAI gene during adenovirus infections.
  • ⁇ HV68 viral miRNAs and pol III transcripts may play a significant role during viral infection, with potential to function as adenovirus VAI RNA and as a unique RNA molecule with unique properties. It has been shown within previous reports that the left end of the viral genome, the area where the ⁇ HV68 pol III transcripts are located, is not required for lytic infection but has important consequences for virus pathogenesis in vivo (Dutia, B. M. et al., J. Gen. Virol. 85:1393-1400 (2004); Clambey, E. T. et al., J. Virol. 76:6532-6544 (2002)).
  • Mouse fibroblast cell lines 3T3 (ATCC CRL-1658) and 3T12 (ATCC CCL-164), as well as the human 293T epithelial cell line were cultured in complete DMEM (Gibco) supplemented with 5% FBS (HyClone), 2mM L-glutamine, 10U/mL penicillin, and lO ⁇ g/mL streptomycin sulfate.
  • SI l tumor cells were generated by Usherwood et. al. (Usherwood, E. J. et al., J. Virol. 70:6516-6518 (1996)).
  • the Sl 1 tumor cell line and A20 lymphoma cell line were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 5OuM ⁇ -mercaptoethanol, ImM sodium pyruvate, 2mM L-glutamine, lOU/mL penicillin, and lO ⁇ g/mL streptomycin sulfate.
  • RPMI 1640 medium Gibco
  • FBS 10OuM ⁇ -mercaptoethanol
  • ImM sodium pyruvate 2mM L-glutamine
  • lOU/mL penicillin lO ⁇ g/mL streptomycin sulfate
  • streptomycin sulfate lO ⁇ g/mL streptomycin sulfate.
  • ⁇ HV68 strain WUMS ATCC VR-1465 was used for all infections. Virus stocks used for infection were passaged, grown, and titer determined on 3Tl 2 cells as previously described (Virgin, H. W
  • RNA isolation was carried out using the rarVanaTM miRNA isolation kit (Ambion, Austin, Tex.), as per manufacturer's instructions. Approximately 5x10 5 to 1x10 6 cells were harvested during each RNA isolation of tissue culture cells.
  • RT-PCR and PCR Amplification were conducted using the primers found in Table 1. RT-PCR amplification reactions were performed in a 25 ⁇ l reaction mix using the OneStep RT-PCR Kit (Qiagen, Valencia, Calif.) containing a final concentration of each primer at 0.5 ⁇ M. 1 O ⁇ l ( ⁇ 100ng) of RNA was treated with RQ 1 RNase-Free DNase (Promega, Madison, Wis.) prior to addition to the RT-PCR reaction mix to eliminate any residual traces of DNA.
  • RT-PCR reactions were performed under the following conditions: 30 min at 50°C (reverse transcription reaction); 15 min at 95°C (heat inactivation of the reverse transcriptase and activation of the Taq polymerase); 40 amplification cycles of 30 sec at 94 0 C followed by 30 sec of 48-56 0 C (dependent on the Tm of the primer set used in a particular reaction) followed by 1 min at 72°C. After the amplification cycles were completed reactions were incubated for an additional 10 min at 72°C.
  • RNA linker oligo was designed to have minimal secondary structure to facilitate ligation by T4 RNA ligase.
  • RNA linker oligo 5'-CUA CGA CAG UCA CCA CCG AUA CCC UGU ACU ACG CAC CAC GAU GAA A-3 ⁇
  • the isolated RNA and RNA linker oligo mixture was combined with 4OU of RNasin (Promega) and 1OU of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) and T4 RNA ligase reaction buffer.
  • the entire reaction mixture was brought to 50 ⁇ l with DEPC treated RNase free water and incubated at 37°C for 1 hour. After ligation, the reaction was stopped by heat inactivating the T4 RNA ligase enzyme by heating the reactions to 70°C for 20 minutes.
  • RNA was isolated from the heat-inactivated ligation reaction mixtures by the addition of 5 ⁇ l of 3M NaOAc pH 5.2 and 125 ⁇ l of 100% ethanol, and incubation of the reactions at -80 0 C for a minimum of 20 minutes. After precipitation, the reactions were spun at top speed (16,170 x g) at 4°C for 15 minutes to pellet the RNA precipitate. The supernatant was removed and the pellet was washed with 200 ⁇ l of 70% ethanol, and spun again for 5 minutes. The 70% ethanol was then removed and the pellet allowed to air dry 2-5 minutes before resuspension in lOO ⁇ l of DEPC treated RNase free water. lO ⁇ l of the ligated RNAs were used within RT-PCR reactions as described above.
  • 5'Monophosphate-Cap-Triphosphate 5'Monophosphate-Cap-Triphosphate Modification Assay.
  • Total RNA was isolated from infected or mock infected cells as described above. In order to remove all 5' phosphate groups from the isolated RNA samples, total RNA was treated with Antarctic Phosphatease as follows: 3 ⁇ g of total RNA was combined with 4OU of RNasin (Promega) and 25U of Antarctic Phosphatase (New England Biolabs) and Antarctic Phosphatase reaction buffer. The entire reaction mixture was brought to 50 ⁇ l with RNase-free water and incubated at 37°C for 1 hour. The phosphatase reaction was stopped by heat inactivating the enzyme at 70 0 C for 10 mins.
  • the Tobacco Acid Pyrophosphatase (TAP) reaction was completed as follows: l ⁇ g of phosphatased RNA was combined with 4OU of RNasin (Promega) and 2.5U of TAP (Eppicentre, Madison, Wis.) and TAP reaction buffer. The entire reaction was brought to 50 ⁇ l with RNase-free water and incubated at 37°C for 90 mins. RNA was isolated as described.
  • the entire amount ( ⁇ 1 ⁇ g) of isolated TAP RNA was used in a RLM-RT-PCR reaction to identify potential 5' cap RNA molecules.
  • the kinase reaction was completed as follows: l ⁇ g of phosphatased RNA was combined with 4OU of RNasin (Promega) and 1OU of T4 Polynucleotide Kinase (New England Biolabs) and 10x T4 DNA Ligase Buffer w/ 1OmM ATP (New England Biolabs). The entire reaction mixture was brought to 50 ⁇ l with RNase free water and incubated at 37°C for 30 mins. The kinase reaction was stopped by heat inactivating the enzyme at 65 0 C for 20 minutes.
  • RNA was isolated from the heat-inactivated reaction as described. The entire amount ( ⁇ 1 ⁇ g) of isolated kinased RNA was used in a RLM-RT-PCR reaction to identify potential 5' non-monophosphate and non-capped RNA molecules.
  • Rapid Amplification of RNA Ends (RARE) Assay To circularize the RNA molecules, 1 ⁇ g of 5'MCT modified RNA was combined with 4OU of RNasin (Promega) and 1OU of T4 RNA ligase (New England Biolabs) and T4 RNA ligase reaction buffer, in the absence of a synthetic RNA linker oligo.
  • RNA isolation and RT-PCR reactions were completed as described.
  • RNA Folding Prediction and RNA Modeling Software All RNA secondary structures depicted were predicted using the software RNAstructure 4.2, created by Mathews, D.H. et. al. (Mathews, D. H. et al., Proc. Natl. Acad. ScL U. S. A 101:7287-7292 (2004)). Predicted structures were modeled into jpeg format using the XRNA software found at http://rna.ucsc.edu/rnacenter/xrna/xrna.html .
  • ⁇ -amanitin treatment of tissue culture cells ⁇ HV68 infected or mock infected 293T cells were treated with either O.Ol ⁇ g/mL, l ⁇ g/mL, or lOO ⁇ g/mL of ⁇ -amanitin (Sigma) over the course of 24 hours to distinguish between the activities of RNA polymerase II and III. 293T cells were infected with ⁇ HV68 for 1 hour prior to the start of the ⁇ -amanitin treatment. After ⁇ -amanitin treatment, total RNA was isolated as described.
  • ⁇ HV68 pol III transcript coding regions Multiple overlapping A box promoter elements are found within the ⁇ HV68 pol III transcript coding regions.
  • ⁇ HV68 encodes eight RNA pol III transcripts previously characterized as vtRNA-like coding sequences (Bowden, R. J. et al., J. Gen. Virol. 78 ( Pt 7):1675-1687 (1997)).
  • Evidence within this work demonstrates that the ⁇ HV68 pol III transcripts do not give rise to RNA molecules resembling the previously proposed vtRNA- like transcripts.
  • These transcripts are herein referred to as viral pol III transcripts ordered 1 through 8 starting from left to right in the ⁇ HV68 genome throughout the example section of the application. All eight of the ⁇ HV68 pol III transcripts encoded by the virus are located within a single locus within the left end of the viral genome between nucleotide 127 and nucleotide 5586.
  • RNA pol III genes are distributed surrounding two open reading frames (ORFs) predicted to be transcribed by RNA pol II, M 1 , a serpin homolog, and M2, a latency associated protein ( Figure I.A.).
  • RNA pol III genes are driven by a variety of different promoter types known as either type 1, 2, or 3.
  • RNA pol III type 1 promoters are internal promoters associated with 5 S rRNA transcription
  • type 2 promoters are also internal promoters associated with tRNA and AIu repeat element transcription
  • type 3 promoters are external promoters associated with U6 snRNA transcription (Paule, M. R. et al., Nucleic Acids Res. 28:1283-1298 (2000); Rowold, D. J.
  • RNA pol III type 2 promoter The bipartite RNA pol III type 2 promoter consists of two separate elements known as the A box and the B box. These internal promoters drive transcription beginning approximately 7 nucleotides upstream from the A box promoter element and are spaced about 21-35 nucleotides apart from each other (Hamada, M. et al., MoI. Cell Biol.
  • the tRNAscan-SE scanning software predicts a start site of transcription utilizing A box-1, the left-most positioned A box promoter element, to drive transcription of these transcripts.
  • a box-1 is used and transcription assumed to proceed to the first run of four or more T residues
  • the predicted secondary structures contain a characteristic tRNA- like fold at the 5' end of the RNA molecule followed by two stem-loop structures that contain one or more miRNAs ( Figure I.D.).
  • the pol III-4 transcript predicted to encode both the miR-Ml -5 and miR-Ml -6 miRNAs, was drawn to include the miR-Ml -6 miRNA gene and terminate transcription at the second canonical RNA pol III transcription termination site.
  • Gammaherpesviruses are known to establish latent infection in lymphocytes and to cause acute/lytic infection within epithelial and fibroblast cells.
  • Latent infection is characterized by limited viral transcription and a lack of viral replication, whereas lytic infection is defined as high levels of viral transcription accompanied by active viral replication.
  • ⁇ HV68 latently infected lymphocyte cells can spontaneously reactivate to lytic replication. This process can be induced in vitro by chemical stimulation of a ⁇ HV68 infected B cell lymphoma cell line.
  • ⁇ HV68 miRNA primary transcripts are transcribed by RNA polymerase III.
  • ⁇ HV68 pol III transcripts predicted to be the pri-miRNA transcripts of ⁇ HV68, were indeed transcribed via RNA polymerase III, ⁇ -amanitin treated 293T cells were infected with ⁇ HV68 and performed RT-PCR analysis to check for the presence of ⁇ HV68 pol IH-I, 4, and 5 transcripts, ⁇ -amanitin is often used to distinguish between the transcriptional activities of RNA polymerases I, II, and III.
  • RNA pol II transcriptional activity is inhibited at low doses of ⁇ -amanitin treatment. Treating 293 T cells with lOO ⁇ g/mL of ⁇ -amanitin for 24 hours completely abolishes RNA polymerase II activity (lack of GAPDH transcription, Figure 3. A.). This same level and duration of ⁇ -amanitin treatment did not inhibit transcription of RNA polymerase III genes as shown by the constant level of tRNA Tyr transcription across all ⁇ -amanitin treatment concentrations ( Figure 3.A.).
  • RNA polymerase II genes RNA polymerase II genes
  • RNA polymerase III genes RNA polymerase III genes.
  • RT-PCR analysis of total RNA isolated from infected 293T cells treated with ⁇ -amanitin using primers specific for the predicted ⁇ HV68 pol III transcripts revealed moderate inhibition of transcription of the ⁇ HV68 pol IH-4 transcript, and no inhibition of transcription of the ⁇ HV68 pol III-5 transcript in contrast to the complete inhibition of the pol II GAPDH gene.
  • ⁇ HV68 pol HI-I transcript could be produced by RNA pol III transcription
  • primers were designed to detect a minimally sized ⁇ HV68 pol III- 1 transcript that could give rise to a pri-miRNA transcript.
  • This minimal ⁇ HV68 pol IH-I transcript was predicted to contain the sequences from Abox-3 through miR-Ml-1.
  • RT- PCR for this shorter product using the same ⁇ -amanitin treatment conditions as above resulted in the amplification of this shorter RT-PCR product resistant to ⁇ -amanitin treatment in a manner consistent with RNA pol III transcription ( Figure 3. B.).
  • RNA pol III transcription of the pri-miRNA viral transcripts within ⁇ HV68 infection the abundance of maturely processed miR-Ml-1 levels was determined by RLM-RT-PCR analysis of the infected ⁇ -amanitin treated cells to measure miR-Ml-1 levels. Shown in Figure 3.B., the overall levels of mature, processed miR-Ml-1 were unchanged throughout the course of the ⁇ -amanitin treatment suggesting that the ⁇ HV68 pol III transcripts identified are responsible for giving rise to the mature miRNA products.
  • RNA transcripts Two new assays to map RNA transcripts were developed using methods based on RLM-RT-PCR. By mapping RNA transcripts using these methods, much greater sensitivity than northern blotting was achieved, in addition to being able to sequence the PCR products for exact identification of the RNA molecule of interest from a total RNA sample.
  • the first of the two new assays is called the 5'MCT assay (5'-monophosphate, cap, and triphosphate), based on the widely used 5'RACE (Rapid Amplification of cDNA Ends) system used for identification of 5' ends of capped mRNAs.
  • 5'MCT assay 5'-monophosphate, cap, and triphosphate
  • 5'RACE Rapid Amplification of cDNA Ends
  • RNA processing enzymes which include the RNase III class of enzymes including both the Drosha and Dicer miRNA processing enzymes (Bartel, D. P. Cell 116:281 -297(2004)), 5'-caps, the common modification of mRNAs transcribed by RNA polymerase II (Rasmussen, E. B. et al., Proc. Natl. Acad. ScL U. S. A 90:7923-7927 (1993)), and 5' triphosphates, which arise from 5' unmodified RNA transcripts.
  • RNA molecules suitable for use within the RLM-RT-PCR procedure are those that contain 5' monophosphates.
  • the only molecules that can naturally undergo reverse ligation are those processed out of a longer RNA transcript, such as maturely processed miRNAs.
  • 5' monophosphates can be added to RNA molecules that originally contained 5' caps or 5' triphosphates within the original total RNA sample ( Figure 4.A.).
  • RNA molecules can be used within reverse ligation reactions and subsequently used with RT-PCR reactions to both identify the genomic position of the 5' nucleotide of a target RNA molecule as well as determine the 5' end biochemical modification of that RNA molecule, hi order to test the fidelity of this assay, it was applied to map the 5' end of a well-known cellular RNA polymerase II gene, ⁇ - actin, as well as a cellular RNA polymerase III gene, tRNA ⁇ RNA was isolated from the mouse fibroblast cell line 3T3 in the presence or absence of ⁇ HV68 infection.
  • RNA Isolated total RNA was subjected to the 5'MCT enzymatic reactions described in Figure 4.A., followed by RLM-RT-PCR with primers specific to either ⁇ -actin of tRNA Asp .
  • ⁇ -actin mRNA is known to be transcribed by RNA pol ⁇ , is capped and has been mapped previously (Park, D. J. MoI. Biotechnol. 29:39-46 (2005)).
  • RNA polymerase III gene tRNA Asp served as a cellular control transcript that should contain transcripts with both 5' monophosphates and 5' triphosphates. This transcript has been mapped previously for its mature, fully processed tRNA form (GoIl, M. G. et al., Science 311:395-398 (2006)).
  • tRNAs contain a 5' monophosphate due to tRNA maturation that results in cleavage of the nascent transcript by RNase P (Torres-Larios, A. et al., Curr. Opin. Struct. Biol. 16:327-335 (2006); Kikovska, E. et al., Nucleic Acids Res. 33:6920-6930 (2005)).
  • Results from the 5'MCT assay map this transcript to the exact same 5' end as previously described (GoIl, M. G. et al., Science 311 :395-398 (2006)), for a RNA molecule with a 5' monophosphate end, which represents the mature fully processed 5' end of tRNA Asp ( Figure 4.B.).
  • Figure 4.B. there was no PCR amplification for tRNA ⁇ within the TAP reaction condition or the phosphatase negative control conditions.
  • Figure 4.B. somewhat unexpectedly, no PCR amplification within the kinased RNA samples was found that represent the 5' triphosphate condition ( Figure 4.B.).
  • RNA to the mature tRNA form leaves only 5' monophosphates and no 5' triphosphates.
  • the 5'MCT assay was employed to map the ⁇ HV68 pol III transcripts.
  • RT-PCR of the circularized RNA across the 5' / 3' junction of the RNA using inverse primer sets, followed by cloning and sequencing of the RT-PCR product allows for the direct sequence mapping of single RNA molecules rather than independent 5' and 3' identification from a pool of molecules, as is the case with traditional 5' and 3' RACE techniques ( Figure 5.).
  • This assay is sometimes referred to as the RARE assay (Rapid Amplification of RNA Ends).
  • ⁇ HV68 pol IH-I had predominant transcription initiation sites at +18 and +20 nucleotides downstream than the previously predicted transcription initiation site.
  • ⁇ HV68 pol HI-I terminated transcription well upstream from the canonical RNA pol III transcription termination site located at genomic nucleotide positions of 319-322. Instead, two different predominant termination sites were mapped, one at genomic positions 258-259 and one at genomic positions 291-292.
  • RNA pol III can terminate transcription at alternative terminations sites (Gunnery, S. et al., J. MoI. Biol. 286:745-757 (1999)).
  • a similar sequence of TTTAT is found at ⁇ HV68 genomic positions 255-259 and is predicted to serve as a site of alternative transcription termination.
  • a sequence of ATTT is found at ⁇ HV68 genomic positions 293-296 and could be contributing to the termination of the longer ⁇ HV68 pol III-l transcript.
  • ⁇ HV68 pol III-4 had only one predominant PCR product that gave rise to a pol III transcript with an transcription initiation site at +14 that of the original predicted transcript and terminated transcription at the first of the two proximal canonical RNA pol III transcription termination signals.
  • ⁇ HV68 pol III-5 also had two predominant PCR products that both initiated transcription near the original predicted +1 transcription initiation site at positions -3 and -4. As with pol III-l, this transcript appears to use multiple transcription termination sites.
  • One termination site is in the canonical stop site predominantly at genomic position 1788.
  • another termination site containing the same alternative termination sequence of the CELO adenovirus, TTATT is found at genomic positions of 1714 - 1718 and also appears to be utilized as a termination sequence for ⁇ HV68 pol III-5 as defined by a major 3' mapped site at genomic position 1716.
  • TTATT the termination sequence for ⁇ HV68 pol III-5 as defined by a major 3' mapped site at genomic position 1716.
  • the precursor miRNA hairpin sequences were mapped that are the intermediate RNA products that eventually give rise to the mature processed miRNAs.
  • the predicted pre-miRNA hairpin structures are shown in Figure 6.E.
  • Precursor miRNAs Ml-I and Ml-7 make perfect pre-miRNAs for the processing of miRNAs miR-Ml-1, -7-5p, and -7-3p, respectively.
  • the pre-miRNA for miR-Ml-5 excludes one nucleotide at the 5' end of the previously sequenced miRNA products and appears to have a four nucleotide overhang at its 3' end which is unusual for miRNA processing.
  • RNAs with certain 5' end biochemical modifications can map RNA from single RNA molecules, are extremely sensitive and can be used for the direct sequencing of target RNA molecules of interest.
  • RNA linker was contacted with 5 ⁇ g of total RNA isolated from 3T3 mouse fibroblast cells. The samples were run on a 15% 7M urea denaturing polyacrylamide gel and stained with ethidium bromide. Results are shown in Figure 11.
  • Example 4 A synthetic control reaction was run to explore the effects various 5'end modifications on the ligation of a 5'OH RNA linker.
  • a population or RNAs with various 5'end modifications were treated with a ligase and ATP.
  • Samples were run on a 15% 7M Urea denaturing polyacrylamide gel and stained with ethidium bromide. Results are shown in Figure 12.
  • Example 5
  • Example 6 A synthetic control reaction was run to explore the effects of various 5'end modifications on the ligation of 5'OH RNA linker in conjunction with phosphatase and kinase treatment. Briefly, a population of RNAs with various 5'end modifications were treated with Antarctic phosphatase then T4 polynucleotide kinase followed by treatment with a 5'OH RNA linker, ligase and ATP. Samples were run on a 15% 7M Urea denaturing polyacrylamide gel and stained with ethidium bromide. Results are shown in Figure 15.

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Abstract

L'invention porte sur des procédés pour déterminer des modifications de produits de transcription d'ARN cellulaire et/ou cartographier les extrémités 5' et 3' de produits de transcription de l'ARN.
PCT/US2008/069178 2007-07-03 2008-07-03 Procédés et compositions pour identifier des produits de transcription d'arn cellulaire WO2009006584A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2666870A1 (fr) * 2012-05-23 2013-11-27 Pathoquest Procédé de traitement différentiel des teneurs en acide nucléique d'un échantillon, échantillon enrichi, kit et leurs utilisations
US10286084B2 (en) * 2014-02-18 2019-05-14 Duke University Compositions for the inactivation of virus replication and methods of making and using the same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FU H. ET AL.: 'Identification of human fetal liver miRNAs by a novel method' FEBS LETTERS vol. 579, no. 17, July 2005, pages 3849 - 3854 *
HONDA A. ET AL.: 'Identification of the 5' terminal structure of influenza virus genome RNA by a newly developed enzymatic method' VIRUS RESEARCH vol. 55, no. 2, June 1998, pages 199 - 206 *
SUNKAR R. ET AL.: 'Identification and characterization of endogenous small interfering RNAs from rice' NUCLEIC ACIDS RESEARCH vol. 33, no. 14, 02 August 2005, pages 4443 - 4454 *
ZHANG M. ET AL.: 'Identification of the 5' terminal sequence of SAR-55 amd MEX-14 strains of Hepatitis E virus and confirmation that the genome is capped' JOURNAL OF MEDICAL VIROLOGY vol. 65, no. 2, October 2001, pages 293 - 295 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2666870A1 (fr) * 2012-05-23 2013-11-27 Pathoquest Procédé de traitement différentiel des teneurs en acide nucléique d'un échantillon, échantillon enrichi, kit et leurs utilisations
WO2013174857A1 (fr) * 2012-05-23 2013-11-28 Pathoquest Procédé pour le traitement différentiel de teneurs en acide nucléique d'un échantillon, enrichissement d'échantillon, kit et utilisations de celui-ci
US10286084B2 (en) * 2014-02-18 2019-05-14 Duke University Compositions for the inactivation of virus replication and methods of making and using the same

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