CN115125292A - Single-base resolution positioning analysis method for inosine modification in RNA assisted by endonuclease - Google Patents
Single-base resolution positioning analysis method for inosine modification in RNA assisted by endonuclease Download PDFInfo
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Abstract
The invention discloses an endonuclease-assisted single-base resolution positioning method for inosine modification in RNA, belonging to the technical field of biology. According to the invention, 3 '-deoxy-A is used for blocking the end of RNA 3' OH, then hEndoV protein is cut at inosine to generate new RNA 3 'OH, the new 3' OH is subjected to polyuridylation and library construction, and sequencing is carried out to obtain inosine modified site information. The method has the advantages of direct principle, simple and convenient operation, no chemical reaction in the whole process, no degradation of RNA, no need of complex bioinformatics algorithm, and capability of directly obtaining single base resolution positioning information modified by inosine in RNA.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an endonuclease-assisted single-base resolution positioning method for inosine modification in RNA.
Background
RNA is composed of a combination of four typical bases including A, U, G, C, and more than 150 epigenetic modifications with important functions are found on RNA except for four standard base nucleotides. Inosine (Ino) is one of the most important post-transcriptional modifications on mammalian RNA, widely found on mRNA, tRNA, lncRNA, miRNA, and siRNA. Ino is produced by the catalytic aminolytic deamination at adenosine C6 by Adenosine Deaminase (ADAR) family proteins acting on RNA, a transition also known as a-to-Ino RNA editing. 3 ADAR proteins have been identified in mammals, ADAR1, ADAR2 with enzymatic activity and ADAR3 without enzymatic activity.
A-to-Ino editing has important biological functions. Since Ino is recognized as G by cellular machinery as a result of pairing with C during reverse transcription, A-to-Ino editing can alter genetic information post-transcriptionally. A-to-Ino editing on mRNA can result in amino acid changes, splice sites and the creation or deletion of stop codons, resulting in post-translational protein changes that favor protein diversity. Ino on the tRNA is located at position 34 of the anticodon loop, where Ino allows recognition of multiple codons by wobble pairing, enhancing the decoding capacity of the tRNA. mirnas and sirnas are able to regulate target genes in a highly specific manner, and a-to-Ino editing regulates the maturation, structure and function of mirnas and sirnas.
Aberrant levels of a-to-Ino editing have been shown to be associated with a variety of human diseases, including a variety of cancers, neurological and neurodegenerative diseases, psychiatric disorders, and autoimmune diseases, among others. Aberrant editing levels at the A-to-Ino editing sites on some transcripts have been shown to be associated with the mechanisms underlying disease. For example, A-to-Ino editing of the pre-mRNA of the 5 hydroxytryptamine 2C receptor occurs at 5 conserved sites in exon 5 (designated A to E), with increased editing levels at the A site in the brain of a suicidal victim of depression and significantly increased editing levels at the E site in patients with major depression. However, the significance of most A-to-Ino editing sites is unknown, and the accurate identification of the position of A-to-Ino editing in the whole transcriptome is the basis for the deep understanding of the biological function and significance of each site.
At present, the single base positioning method edited by the transcriptome range A-to-Ino is mainly RNA-seq. Ino pairs with C during reverse transcription so that it is read as G during sequencing, and thus can recognize the A-to-Ino editing site based on the A-to-G conversion. Although the principle is simple, in the actual analysis process, due to the interference factors such as alignment error, single nucleotide polymorphism, somatic mutation and the like, the RNA-seq can generate a large number of false positive sites. Although bioinformatic approaches such as statistical modeling, filtering and/or alignment, and integration of other genomic information were developed to equal the reduction of false positives, the accuracy of RNA-seq identification of a-to-Ino editing sites remains poor. Identification of A-to-Ino edits within the transcriptome RangeOne of the chemical methods for the site is "ICE-seq", N produced by the reaction of Ino with acrylonitrile 1 Cyanoethylarinine hinders cDNA extension during reverse transcription, and "erasure" signals generated during sequencing are screened out during library construction due to too short a fragment that produces a truncated cDNA, and can be used to identify a-to-Ino editing sites, but this approach fails to identify 100% edited sites. Another method involving chemical reactions is Ino-specific sequencing, where nuclease T1 is characterized by cleavage of RNA at G or Ino, where G forms a stable adduct with glyoxal in the presence of borate to resist cleavage by nuclease T1, and Ino is cleaved by nuclease T1 because it cannot form a stable adduct. The method comprises the steps of firstly oxidizing and biotinylating the 3' end of RNA through a chemical reaction, then treating and protecting G on the RNA through glyoxal and borate, then fixing the RNA on a streptomycin functional magnetic bead through biotin-streptomycin affinity, cutting the RNA at Ino by nuclease T1, and then performing library construction and sequencing on the cut RNA to analyze the A-to-Ino editing site. The disadvantage of this method is that the procedure is complicated and if 100% protection of G is not achieved, G can also be cleaved by nuclease T1 resulting in false positive sites. Chemical methods have the common disadvantage that severe chemical reactions cause severe degradation of RNA, so mild enzymatic reaction methods should be developed for single base resolution mapping analysis a-to-Ino editing.
hEndoV (human endonuclease V), an endonuclease for Ino on RNA, specifically recognizes and cleaves Ino on RNA, and generates a 3 ' OH and 5 ' phosphate nick at its downstream 3 ' end second phosphodiester bond. Based on the characteristics, the invention provides a single base resolution positioning method of Ino in RNA assisted by endonuclease.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an endonuclease-assisted single-base resolution positioning analysis method for inosine modification in RNA, namely an Ino single-base resolution positioning analysis method which can be applied to the field of RNA modification sequencing, does not need chemical reagent treatment, does not need complex bioinformatics algorithm, and has high sensitivity, high selectivity and simple and convenient operation.
The purpose of the invention is realized by the following technical scheme:
an endonuclease-assisted single-base resolution localization analysis method for inosine modification in RNA comprises the following steps:
(1) fragmenting RNA of a cell or tissue sample;
(2) 3' OH end-blocking of fragmented RNA: adding 3 'deoxy-A to the 3' OH end of the fragmented RNA using polyA polymerase catalyzed 3 'deoxy-ATP to block the 3' OH end of the RNA, followed by purification of the RNA;
(3) cleaving the 3 'OH-end-blocked RNA using the hEndoV protein to generate a new RNA 3' OH at inosine;
(4) 3' polyuridylation of cleaved RNA: catalyzing UTP by using polyU polymerase to add U tail to the 3 'OH end of RNA, and carrying out 3' polyuridylation;
(5) taking 3 'polyuridylated RNA as a template, and carrying out reverse transcription reaction by adopting a reverse transcription primer with polyadenylic acid and a 3' joint to obtain cDNA;
(6) adding a 5 'linker to the cDNA by a DNA polymerase extension reaction using a primer having a 5' linker to obtain a DNA for amplification;
(7) performing PCR amplification by using the DNA as a template, and sequencing an amplified product;
(8) single base recognition with inosine modification: and the second base G from 3 'to 5' after the double-ended joint is removed in the sequencing result is an inosine modified base.
According to the method, Ino is specifically cut by the hEndoV protein, and single-base resolution positioning information of Ino is obtained through sequencing.
The aforementioned cleavage reaction of the hEndoV protein: NEBuffer 4(NEB, cat # B7004) was used as a reaction buffer, and the buffer system of the buffer reaction solution was Tris-CH 3 COOH，Mg 2+ Is 10mM, and a certain amount of the hEndoV protein (determined according to the activity of the actual protein) is used, and the reaction condition is preferably 37 ℃ for 30 min.
The aforementioned cleavage reaction of the hENDOV protein refers to the specific cleavage reaction of the hENDOV protein to Ino on RNA.
The invention has the following advantages and beneficial effects:
1) the invention has direct principle, simple method and no need of complex pretreatment.
2) The invention does not need chemical reaction to process RNA and can not cause violent degradation of RNA.
3) The hENDOV protein cleavage reaction can be completed in only 30 minutes, and the whole experiment time is greatly shortened.
4) The cleavage reaction involved in the invention has high specificity (only Ino is cleaved, and A is not cleaved) and high efficiency (complete cleavage), and is beneficial to positioning analysis.
5) The RNA library construction method does not need single-strand ligation reaction, has high library construction efficiency, and is beneficial to sequencing and site analysis.
6) The invention does not relate to a complex bioinformatics algorithm and is beneficial to the identification of the A-to-Ino editing sites.
Drawings
FIG. 1 is a schematic diagram of a single base resolution mapping analysis method for inosine modification in RNA assisted by endonuclease according to the present invention. The 3 'OH end of RNA was first blocked by 3' -deoxy-a, then a new 3 'OH was generated at the 3' second phosphodiester bond of Ino by hEndoV protein cleavage, the newly generated 3 'OH was subsequently 3' polyuridylated, and single base resolution mapping analysis of Ino was achieved via library construction and sequencing.
FIG. 2 is a PAGE gel of the hEndoV protein of the present invention specifically cleaving an artificially synthesized RNA containing an Ino modification. (A) Schematic representation of the Ino modification on the hEndoV cleavage RNA; (B) the cleavage effect of the hAndoV protein was evaluated using synthetic RNA strands containing Ino modification (Ino-RNA-1 and Ino-RNA-2, see Table 2 for sequence) as substrates, and (C) the cleavage specificity of the hAndoV protein was evaluated using RNA strands not containing Ino modification (A-RNA-1, see Table 2 for sequence) as substrates.
FIG. 3 is a diagram showing the sequencing effect of single-base resolution mapping analysis of inosine modification on a synthetic 45nt Ino-RNA-1 oligonucleotide chain in the present invention.
Detailed Description
In some embodiments, the method for single base resolution mapping analysis of inosine modification in endonuclease-assisted RNA of the present invention specifically comprises the following steps:
(1) fragmenting; taking 5 μ g of mRNA extracted from tissue or cells of a biological sample, using NEBNext RNA Mg ++ The fragmentation module (NEB, cat # E6150) fragments the mRNA to around 1000nt in length.
(2) 3' OH end blocking of the above RNA: to the above mRNA were added 5U of polyA polymerase (NEB, cat # M0276), 1 XPolyA reaction buffer (dispensed with the enzyme), 1mM 3' deoxy-ATP (Sigma-Aldrich, cat # C9137) and 20U of RNase inhibitor, reacted at 37 ℃ for 30min, and then RNA was purified using RNA Clean & concentrate Kits (ZYMO RESEARCH, cat # R1013) (10. mu.L eluted).
(3) The above RNA was subjected to hEndoV cleavage: to the above 10. mu.L of RNA were added a certain amount of hEndoV protein, 1 XNEBuffer 4 buffer (NEB, cat. No. B7004) and 20U of RNase inhibitor to a final volume of 20. mu.L, and cleavage reaction was carried out at 37 ℃ for 30 min.
The expression process of the hENDOV protein is as follows, wherein the full-length coding sequence (shown in table 1) of the hENDOV is inserted into a pET-28a (+) plasmid to obtain a hENDOV-pET-28a (+) recombinant plasmid, and the hENDOV-pET-28a (+) recombinant plasmid is transformed into escherichia coli BL21(DE 3); the conditions for inducing protein expression are that the IPTG concentration is 1mM, the induction temperature is 25 ℃, and the induction time is 10 h; the protein was purified using a Beyogold His-tag Purification Resin (reduction-resistant chelating type) (Biyuntian Biotechnology, cat # P2210). The procedure for assessing the activity of the hAndoV protein was as follows, and a total of 10. mu.L of a reaction system containing 1. mu.M of RNA (Ino-RNA-1, Ino-RNA-2 and A-RNA-1, see Table 2 for sequence), a certain amount of hAndoV, 20U of RNase inhibitor and 1 XNEBuffer 4 buffer (NEB, cat # B7004) was incubated at 37 ℃ for 30 minutes, and 1. mu.L of 10 Xloading buffer (Takara, cat # 9157) was added to the system to terminate the reaction, and the reaction results were confirmed by 20% PAGE (FIG. 2).
(4) 3' polyuridylation of the above RNA: to the above 20. mu.L reaction system were added 1U of polyU polymerase (NEB, cat. No. M0337), 1 XNEBuffer 2 buffer (dispensed with the enzyme), 100. mu.M UTP and 20U of RNase inhibitor to a final volume of 40. mu.L. This system was run at 37 ℃ for 8min and RNA was purified using RNA Clean & concentration Kits (ZYMO RESEARCH, cat. No. R1013) (20. mu.L elution).
(5) The RNA is subjected to reverse transcription to synthesize cDNA: a30. mu.L reverse transcription system consisted of 20. mu.L of the above RNA, 200UM-MuLV reverse transcriptase (Bio/industry, cat # B500517), 1 XT reaction buffer (distributed with the enzyme), 0.33. mu.M 3' adaptor-A 20 NC primers (sequence shown in Table 2), 0.5mM dNTP and 20U RNase inhibitor, and this system was reacted at 42 ℃ for 90 min.
(6) RNA removal and cDNA purification: to the reverse transcription reaction (30. mu.L) was added 1. mu.L exonuclease I (NEB, cat # M0293), 5. mu.L exonuclease I reaction buffer (dispensed with the enzyme), and 14. mu.L L H 2 O, reacting at 37 ℃ for 30min to remove excess reverse transcription primer; then, 10. mu.L NaOH (1M) and 10. mu.L EDTA (0.5M) were added to the exonuclease I reaction system (50. mu.L), and reacted at 65 ℃ for 15min to remove RNA in the system, using Oligo Clean&The cDNA was recovered from the Concentrator Kits (ZYMO RESEARCH, cat # D4060) (10. mu.L of elution).
(7) Extension of the cDNA above 5' linker was added: the reaction system (30. mu.L) consisted of 10. mu.L of the above cDNA, 5U of Bsu DNA polymerase (NEB, cat # M0330), 1 XNEBuffer 2 buffer (dispensed with the enzyme), 0.33. mu.M of 5' adaptor-N 6 Primers (sequences are shown in Table 2), 1mM dNTP, and the reaction is carried out at 37 ℃ for 1 h.
(8) DNA purification: mu.L of exonuclease I (NEB, cat # M0293), 5. mu.L of exonuclease I reaction buffer (dispensed with enzyme), and 14. mu. L H were added to the precursor system (30. mu.L) 2 O, reacting at 37 ℃ for 30min to remove excessive 5' adaptor-N 6 Primer, Oligo Clean is adopted&The DNA was recovered from the Concentrator Kits (ZYMO RESEARCH, cat. No. D4060) (15. mu.L of the eluate).
(9) The above DNA was subjected to PCR amplification: the PCR system (50. mu.L) consisted of 15. mu.L of the above DNA, 2 Xamplification buffer (holy next, cat. 10136ES01), 0.5. mu. M P5 and P7 primer (sequence see Table 2). Amplification cycle time program: firstly, denaturation is carried out for 1min at 98 ℃; ② denaturation at 98 ℃ for 10 sec; ③ annealing at 60 ℃ for 25 sec; extension at 72 ℃ for 50 sec; the fourth step is repeated for 30 times; extension at 72 ℃ for 10min and storage at 4 ℃.
(10) The resulting amplification product was sequenced.
TABLE 1 full length coding sequence of hEndoV
TABLE 2 oligonucleotide chains referred to in the embodiments
The following examples are intended to further illustrate the present invention and should not be construed as limiting the present invention, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and shall be included within the scope of the present invention.
Unless otherwise specified, the technical means used in the examples include nucleic acid extraction, kit purification, reverse transcription, polymerase chain reaction, and the like, which are conventional means well known to those skilled in the art.
Example 1: standard synthetic RNA analysis
(1) 100ng of artificially synthesized Ino-RNA-1 strand (see the sequence in Table 3), 5U of polyA polymerase, 1mM of 3' deoxy-ATP, 1 XpolyA reaction buffer and 20U of RNase inhibitor were added to a 10. mu.L reaction system, and the RNA was purified by using a kit after reaction at 37 ℃ for 30 min.
(2) The RNA was subjected to the hEndoV protein cleavage reaction. To the above RNA were added an amount of the hENDOV protein, 1 XNEBuffer 4 and 20U of RNase inhibitor to make the final volume 20. mu.L, and the cleavage reaction was performed at 37 ℃ for 30 min.
(3) The RNA was subjected to polyuridine formation reaction. To the above 20. mu.L reaction system were added 1U of polyU polymerase, 100. mu.M UTP, 1 XNEBuffer 2 and 20U of RNase inhibitor to a final volume of 40. mu.L. The system was purified for RNA using a kit after 8min at 37 ℃.
(4) The RNA is subjected to reverse transcription. The reverse transcription system (30. mu.L) included 10. mu.L of the above RNA, 0.33. mu.M of 3' adaptor-A 20 GC primers (sequence shown in Table 3), 0.5mM dNTP, 200U reverse transcriptase, 1 × reverse transcription buffer, 20U RNase inhibitor, the system is reacted at 42 ℃ for 90 min. Since the standard synthetic RNA analysis system using Ino-RNA-1 is simple, the step of recovering cDNA here is simplified to add 1. mu.L of ribonuclease A to the reaction-terminated reverse transcription system, react at 37 ℃ for 30min to remove RNA, and recover cDNA by means of a kit.
(5) The cDNA was subjected to 5' linker addition reaction. The 5 'linker addition reaction system (30. mu.L) comprising 10. mu.L of the above cDNA, 5U Bsu DNA polymerase, 0.33. mu.M 5' adaptor primer (see sequence in Table 3), 1mM dNTP, 1 XNEBuffer 2 was performed at 37 ℃ for 1 hour.
(6) And (3) taking the reaction DNA to carry out polymerase chain amplification reaction. A25. mu.L PCR reaction was carried out using 2. mu.L of the above DNA, 12.5. mu.L of 2 Xamplification buffer, 10. mu. M P5, and 1.25. mu.L and 8. mu. L H of each of the P7 primers (see Table 3 for sequences) 2 And (C) O. Amplification cycle time program: firstly, denaturation is carried out for 1min at 98 ℃; ② denaturation at 98 ℃ for 10 sec; ③ annealing at 60 ℃ for 25 sec; extension at 72 ℃ for 50 sec; the fourth step is repeated for 30 times; extension at 72 ℃ for 10min and storage at 4 ℃. Samples were directly subjected to Sanger sequencing, the sequencing results are shown in FIG. 3, where the second base 3 'to 5' after removal of the linker is G (boxed label), which is the inosine modification in the original Ino-RNA-1.
TABLE 3 oligonucleotide chains referred to in example 1
Sequence listing
<110> Wuhan university
<120> endonuclease-assisted single-base resolution positioning analysis method for inosine modification in RNA
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 846
<212> DNA
<213> Homo sapiens
<400> 1
atggccctgg aggcggcggg agggccgccg gaggaaacgc tgtcactgtg gaaacgggag 60
caagctcggc tgaaggccca cgtcgtagac cgggacaccg aggcgtggca gcgagacccc 120
gccttctcgg gtctgcagag ggtcgggggc gttgacgtgt ccttcgtgaa aggggacagt 180
gtccgcgctt gtgcttccct ggtggtgctc agcttccctg agctcgaggt ggtgtatgag 240
gagagccgca tggtcagcct cacagccccc tacgtgtcgg gcttcctggc cttccgagag 300
gtgcccttct tgctggagct ggtgcagcag ctgcgggaga aggagccggg cctcatgccc 360
caggtccttc ttgtggatgg aaacggggta ctccaccacc gaggctttgg ggtggcctgc 420
caccttggcg tccttacaga cctgccgtgt gttggggtgg ccaagaaact tctgcaggtg 480
gatgggctgg agaacaacgc cctgcacaag gagaagatcc gactcctgca gactcgagga 540
gactcattcc ctctgctggg agactctggg actgtcctgg gaatggccct gaggagccac 600
gaccgcagca ccaggcccct ctacatctcc gtgggccaca ggatgagcct ggaggccgct 660
gtgcgcctga cttgctgctg ctgcaggttc cggatcccag agcccgtgcg ccaggctgac 720
atctgctccc gagagcacat ccgcaagtcg ctgggactcc ccgggccacc cacaccgagg 780
agcccgaagg cgcagaggcc agtggcatgc cccaaaggag actccggaga gtcctcagca 840
ctttgt 846
Claims (5)
1. An endonuclease-assisted single-base resolution localization analysis method for inosine modification in RNA is characterized in that: the method comprises the following steps:
(1) fragmenting RNA of a cell or tissue sample;
(2) 3' OH end-capping the fragmented RNA;
(3) cleaving the 3' OH-terminated blocked RNA using the hEndoV protein;
(4) 3' polyuridylation is carried out on the cut RNA;
(5) taking 3 'polyuridylated RNA as a template, and carrying out reverse transcription reaction by adopting a reverse transcription primer with polyadenyle and a 3' joint to obtain cDNA;
(6) adding a 5 'linker to the cDNA by a DNA polymerase extension reaction using a primer having a 5' linker to obtain a DNA for amplification;
(7) performing PCR amplification by using the DNA as a template, and sequencing an amplified product;
(8) single base recognition with inosine modification: and the second base G from 3 'to 5' after the double-ended joint is removed in the sequencing result is an inosine modified base.
2. The method for single base resolution mapping analysis of inosine modification in RNA assisted by endonuclease according to claim 1, wherein: the step (2) is as follows: polyA polymerase is used to catalyze 3 ' deoxy-ATP addition of 3 ' deoxy-a to the 3 ' OH end of fragmented RNA.
3. The method for single base resolution mapping analysis of inosine modification in RNA assisted by endonuclease according to claim 1, wherein: in the step (3), the reaction buffer for RNA cleavage using the hENDOV protein was Tris-CH 3 COOH is a buffer system in which Mg is present 2+ Is 10 mM.
4. The method for single base resolution mapping analysis of inosine modification in RNA supported by endonuclease according to claim 3, wherein: the reaction conditions for RNA cleavage were 37 ℃ for 30 min.
5. The method for single base resolution mapping analysis of inosine modification in RNA assisted by endonuclease according to claim 1, wherein: the step (4) is as follows: UTP was catalyzed by polyU polymerase to add a U-tail to the 3 'OH terminus of RNA for 3' polyuridylation.
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