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Transcriptome-wide identification of adenosine-to-inosine editing using the ICE-seq method

Abstract

Inosine (I), a modified base found in the double-stranded regions of RNA in metazoans, has various roles in biological processes by modulating gene expression. Inosine is generated from adenosine (A) catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes in a process called A-to-I RNA editing. As inosine is converted to guanosine (G) by reverse transcription, the editing sites can be identified by simply comparing cDNA sequences with the corresponding genomic sequence. One approach to screening I sites is by deep sequencing based on A-to-G conversion from genomic sequence to cDNA; however, this approach produces a high rate of false positives because it cannot efficiently eliminate G signals arising from inevitable mapping errors. To address this issue, we developed a biochemical method to identify inosines called inosine chemical erasing (ICE), which is based on cyanoethylation combined with reverse transcription. ICE was subsequently combined with deep sequencing (ICE-seq) for the reliable identification of transcriptome-wide A-to-I editing sites. Here we describe a protocol for the practical application of ICE-seq, which can be completed within 22 d, and which allows the accurate identification of transcriptome-wide A-to-I RNA editing sites.

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Figure 1: Chemistry and outline of ICE-seq.
Figure 2: Data processing flow of ICEBreaker.
Figure 3: Evaluation of cyanoethylation using the ICE method.
Figure 4: Genome-wide views of the regions with editing sites piled with the mapped reads from ICE-seq.
Figure 5: Identification of A-to-I RNA editing sites using the ICE score.

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References

  1. Bjork, G. Biosynthesis and function of modified nucleosides. in tRNA: Structure, Biosynthesis, and Function (eds. Soll, D., Rajbandhary, U. and T.L. Rajbandhary) 165–205 (American Society for Microbiology, 1995).

  2. Grosjean, H. Modification and editing of RNA: historical overview and important facts to remember. in Topics in Current Genetics vol. 12, 1–22 (Springer-Verlag, 2005).

    Article  CAS  Google Scholar 

  3. Suzuki, T. Biosynthesis and function of tRNA wobble modifications. in Topics in Current Genetics vol. 12, 24–69 (Springer-Verlag, 2005).

    Google Scholar 

  4. Nishikura, K. Functions and regulation of RNA editing by ADAR deaminases. Annu. Rev. Biochem. 79, 321–349 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Kim, U. et al. Purification and characterization of double-stranded RNA adenosine deaminase from bovine nuclear extracts. J. Biol. Chem. 269, 13480–13489 (1994).

    PubMed  CAS  Google Scholar 

  6. Hough, R.F. & Bass, B.L. Purification of the Xenopus laevis double-stranded RNA adenosine deaminase. J. Biol. Chem. 269, 9933–9939 (1994).

    PubMed  CAS  Google Scholar 

  7. Kim, U., Wang, Y., Sanford, T., Zeng, Y. & Nishikura, K. Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing. Proc. Natl. Acad. Sci. USA 91, 11457–11461 (1994).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Melcher, T. et al. A mammalian RNA editing enzyme. Nature 379, 460–464 (1996).

    Article  PubMed  CAS  Google Scholar 

  9. Higuchi, M. et al. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406, 78–81 (2000).

    Article  PubMed  CAS  Google Scholar 

  10. Wang, Q., Khillan, J., Gadue, P. & Nishikura, K. Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 290, 1765–1768 (2000).

    Article  PubMed  CAS  Google Scholar 

  11. Wang, Q. et al. Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. J. Biol. Chem. 279, 4952–4961 (2004).

    Article  PubMed  CAS  Google Scholar 

  12. Jepson, J.E. & Reenan, R.A. RNA editing in regulating gene expression in the brain. Biochim. Biophys. Acta 1779, 459–470 (2008).

    Article  PubMed  CAS  Google Scholar 

  13. Tonkin, L.A. et al. RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans. EMBO J. 21, 6025–6035 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Levanon, E.Y. et al. Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nat. Biotechnol. 22, 1001–1005 (2004).

    Article  PubMed  CAS  Google Scholar 

  15. Sakurai, M. et al. A biochemical landscape of A-to-I RNA editing in the human brain transcriptome. Genome Res. 24, 522–534 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Barak, M. et al. Evidence for large diversity in the human transcriptome created by Alu RNA editing. Nucleic Acids Res. 37, 6905–6915 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Paz-Yaacov, N. et al. Adenosine-to-inosine RNA editing shapes transcriptome diversity in primates. Proc. Natl. Acad. Sci. USA 107, 12174–12179 (2010).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. Tojo, K. et al. Dystonia, mental deterioration, and dyschromatosis symmetrica hereditaria in a family with ADAR1 mutation. Mov. Disord. 21, 1510–1513 (2006).

    Article  PubMed  Google Scholar 

  19. Rice, G.I. et al. Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature. Nat. Genet. 44, 1243–1248 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Maas, S., Patt, S., Schrey, M. & Rich, A. Underediting of glutamate receptor GluR-B mRNA in malignant gliomas. Proc. Natl. Acad. Sci. USA 98, 14687–14692 (2001).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Kawahara, Y. et al. Glutamate receptors: RNA editing and death of motor neurons. Nature 427, 801 (2004).

    Article  PubMed  CAS  Google Scholar 

  22. Higuchi, M. et al. RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency. Cell 75, 1361–1370 (1993).

    Article  PubMed  CAS  Google Scholar 

  23. Burns, C.M. et al. Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387, 303–308 (1997).

    Article  PubMed  CAS  Google Scholar 

  24. Hoopengardner, B., Bhalla, T., Staber, C. & Reenan, R. Nervous system targets of RNA editing identified by comparative genomics. Science 301, 832–836 (2003).

    Article  PubMed  CAS  Google Scholar 

  25. Ohlson, J., Pedersen, J.S., Haussler, D. & Ohman, M. Editing modifies the GABA(A) receptor subunit α3. RNA 13, 698–703 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Rueter, S.M., Dawson, T.R. & Emeson, R.B. Regulation of alternative splicing by RNA editing. Nature 399, 75–80 (1999).

    Article  PubMed  CAS  Google Scholar 

  27. Chen, L.L., DeCerbo, J.N. & Carmichael, G.G. Alu element-mediated gene silencing. EMBO J. 27, 1694–1705 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Sakurai, M., Yano, T., Kawabata, H., Ueda, H. & Suzuki, T. Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome. Nat. Chem. Biol. 6, 733–740 (2010).

    Article  PubMed  CAS  Google Scholar 

  29. Agranat, L., Raitskin, O., Sperling, J. & Sperling, R. The editing enzyme ADAR1 and the mRNA surveillance protein hUpf1 interact in the cell nucleus. Proc. Natl. Acad. Sci. USA 105, 5028–5033 (2008).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Bass, B.L. How does RNA editing affect dsRNA-mediated gene silencing? Cold Spring Harb. Symp. Quant. Biol. 71, 285–292 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Hundley, H.A., Krauchuk, A.A. & Bass, B.L. C. elegans and H. sapiens mRNAs with edited 3′ UTRs are present on polysomes. RNA 14, 2050–2060 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Borchert, G.M. et al. Adenosine deamination in human transcripts generates novel microRNA binding sites. Hum. Mol. Genet. 18, 4801–4807 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Wang, Q. et al. ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding. RNA 19, 1525–1536 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Kawahara, Y., Zinshteyn, B., Chendrimada, T.P., Shiekhattar, R. & Nishikura, K. RNA editing of the microRNA-151 precursor blocks cleavage by the Dicer-TRBP complex. EMBO Rep. 8, 763–769 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Kawahara, Y. et al. Redirection of silencing targets by adenosine-to-inosine editing of miRNAs. Science 315, 1137–1140 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Kawahara, Y. Quantification of adenosine-to-inosine editing of microRNAs using a conventional method. Nat. Protoc. 7, 1426–1437 (2012).

    Article  PubMed  CAS  Google Scholar 

  37. Paz, N. et al. Altered adenosine-to-inosine RNA editing in human cancer. Genome Res. 17, 1586–1595 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Li, M. et al. Widespread RNA and DNA sequence differences in the human transcriptome. Science 333, 53–58 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Bahn, J.H. et al. Accurate identification of A-to-I RNA editing in human by transcriptome sequencing. Genome Res. 22, 142–150 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Peng, Z. et al. Comprehensive analysis of RNA-seq data reveals extensive RNA editing in a human transcriptome. Nat. Biotechnol. 30, 253–260 (2012).

    Article  PubMed  CAS  Google Scholar 

  41. Kleinman, C.L. & Majewski, J. Comment on ‘Widespread RNA and DNA sequence differences in the human transcriptome’. Science 335, 1302 (2012).

    Article  PubMed  CAS  Google Scholar 

  42. Lin, W., Piskol, R., Tan, M.H. & Li, J.B. Comment on ‘Widespread RNA and DNA sequence differences in the human transcriptome’. Science 335, 1302 (2012).

    Article  PubMed  CAS  Google Scholar 

  43. Pickrell, J.K., Gilad, Y. & Pritchard, J.K. Comment on ‘Widespread RNA and DNA sequence differences in the human transcriptome’. Science 335, 1302 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Piskol, R., Peng, Z., Wang, J. & Li, J.B. Lack of evidence for existence of noncanonical RNA editing. Nat. Biotechnol. 31, 19–20 (2013).

    Article  PubMed  CAS  Google Scholar 

  45. Sakurai, M. & Suzuki, T. Biochemical identification of A-to-I RNA editing sites by the inosine chemical erasing (ICE) method. Methods Mol. Biol. 718, 89–99 (2011).

    Article  PubMed  CAS  Google Scholar 

  46. Sherry, S.T. et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 29, 308–311 (2001).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Morse, D.P. & Bass, B.L. Detection of inosine in messenger RNA by inosine-specific cleavage. Biochemistry 36, 8429–8434 (1997).

    Article  PubMed  CAS  Google Scholar 

  48. Cattenoz, P.B., Taft, R.J., Westhof, E. & Mattick, J.S. Transcriptome-wide identification of A>I RNA editing sites by inosine specific cleavage. RNA 19, 257–270 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Ota, H. et al. ADAR1 forms a complex with dicer to promote microRNA processing and RNA-induced gene silencing. Cell 153, 575–589 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Breslauer, K.J., Frank, R., Blocker, H. & Marky, L.A. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746–3750 (1986).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  51. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Smith, T.F. & Waterman, M.S. Identification of common molecular subsequences. J. Mol. Biol. 147, 195–197 (1981).

    Article  PubMed  CAS  Google Scholar 

  53. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Metzker, M.L. Sequencing technologies - the next generation. Nat. Rev. Genet. 11, 31–46 (2010).

    Article  PubMed  CAS  Google Scholar 

  55. Martin, J.A. & Wang, Z. Next-generation transcriptome assembly. Nat. Rev. Genet. 12, 671–682 (2011).

    Article  PubMed  CAS  Google Scholar 

  56. Sims, D., Sudbery, I., Ilott, N.E., Heger, A. & Ponting, C.P. Sequencing depth and coverage: key considerations in genomic analyses. Nat. Rev. Genet. 15, 121–132 (2014).

    Article  PubMed  CAS  Google Scholar 

  57. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  58. Green, M.R. & Sambrook, J. Molecular Cloning: a Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2012).

  59. Li, J.B. et al. Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science 324, 1210–1213 (2009).

    Article  PubMed  CAS  Google Scholar 

  60. Genomes Project, C. et al. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).

    Article  CAS  Google Scholar 

  61. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Huang, S. et al. SOAPsplice: genome-wide ab initio detection of splice junctions from RNA-seq data. Front. Genet. 2, 46 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Garber, M., Grabherr, M.G., Guttman, M. & Trapnell, C. Computational methods for transcriptome annotation and quantification using RNA-seq. Nat. Methods 8, 469–477 (2011).

    Article  PubMed  CAS  Google Scholar 

  64. Wu, T.D. & Watanabe, C.K. GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21, 1859–1875 (2005).

    Article  PubMed  CAS  Google Scholar 

  65. Kent, W.J. BLAT: the BLAST-like alignment tool. Genome Res. 12, 656–64 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Cibulskis, K. et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 31, 213–219 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Koboldt, D.C. et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 22, 568–576 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank members of the Suzuki laboratory for their experimental assistance and fruitful discussions of this study. Special thanks are due to A. Fujiyama and A. Toyoda (National Institute of Genomics, Japan) for deep sequencing. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the New Energy and Industrial Technology Development Organization (to T.S.).

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T.S. and M.S. designed a protocol for the biochemical part. H.U. developed all computational methods and wrote a protocol for in silico analyses. All authors wrote the manuscript. T.S. supervised the study.

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Correspondence to Tsutomu Suzuki.

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Suzuki, T., Ueda, H., Okada, S. et al. Transcriptome-wide identification of adenosine-to-inosine editing using the ICE-seq method. Nat Protoc 10, 715–732 (2015). https://doi.org/10.1038/nprot.2015.037

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