Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome

Nature Chemical Biology volume 6, pages 733–740 (2010)Cite this article

Subjects

Abstract

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional processing event involved in diversifying the transcriptome responsible for various biological processes. Although bioinformatic approaches have predicted a number of A-to-I editing sites in cDNAs, the human transcriptome is thought to still harbor large numbers of as-yet-unknown editing sites. Exploring new editing sites requires a biochemical method to accurately identify inosines on RNA strands. We here describe a chemical method to identify inosines, called inosine chemical erasing (ICE), that is based on cyanoethylation combined with reverse transcription. We carried out a large-scale verification of the ICE method focusing on 642 regions in human cDNA and identified 5,072 editing sites, including 4,395 new sites. Functional study revealed that A-to-I intronic editing in the SARS gene mediated by ADAR1 is involved in preventing aberrant exonization of Alu sequence into mature mRNA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cyanoethylation of inosine and outline of the ICE method.
Figure 2: Inosine-specific cyanoethylation and detection of ce1I by primer extension.
Figure 3: Inosines erased in the sequence chromatograms.
Figure 4: Inosine-specific cyanoethylation affects cDNA amplification.
Figure 5: Identification of A-to-I editing sites in human brain RNAs by the ICE method.
Figure 6: Downregulation of ADAR1 results in aberrant exonization of the Alu sequence in the SARS gene.

Similar content being viewed by others

References

  1. Bass, B.L. RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846 (2002).

    Article  CAS  Google Scholar 

  2. 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  CAS  Google Scholar 

  3. 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  CAS  Google Scholar 

  4. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Maas, S., Kawahara, Y., Tamburro, K.M. & Nishikura, K. A-to-I RNA editing and human disease. RNA Biol. 3, 1–9 (2006).

    Article  CAS  Google Scholar 

  7. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Levanon, E.Y. et al. Evolutionarily conserved human targets of adenosine to inosine RNA editing. Nucleic Acids Res. 33, 1162–1168 (2005).

    Article  CAS  Google Scholar 

  10. 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  CAS  Google Scholar 

  11. Kim, D.D. et al. Widespread RNA editing of embedded alu elements in the human transcriptome. Genome Res. 14, 1719–1725 (2004).

    Article  CAS  Google Scholar 

  12. Athanasiadis, A., Rich, A. & Maas, S. Widespread A-to-I RNA editing of Alu-containing mRNAs in the human transcriptome. PLoS Biol. 2, e391 (2004).

    Article  Google Scholar 

  13. Levanon, K., Eisenberg, E., Rechavi, G. & Levanon, E.Y. Letter from the editor: Adenosine-to-inosine RNA editing in Alu repeats in the human genome. EMBO Rep. 6, 831–835 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Osenberg, S., Dominissini, D., Rechavi, G. & Eisenberg, E. Widespread cleavage of A-to-I hyperediting substrates. RNA 15, 1632–1639 (2009).

    Article  CAS  Google Scholar 

  22. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Nishikura, K. Editor meets silencer: crosstalk between RNA editing and RNA interference. Nat. Rev. Mol. Cell Biol. 7, 919–931 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Nishimoto, Y. et al. Determination of editors at the novel A-to-I editing positions. Neurosci. Res. 61, 201–206 (2008).

    Article  CAS  Google Scholar 

  28. Yoshida, M. & Ukita, T. Modification of nucleosides and nucleotides. VII. Selective cyanoethylation of inosine and pseudouridine in yeast transfer ribonucleic acid. Biochim. Biophys. Acta 157, 455–465 (1968).

    Article  CAS  Google Scholar 

  29. Mengel-Jørgensen, J. & Kirpekar, F. Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry. Nucleic Acids Res. 30, e135 (2002).

    Article  Google Scholar 

  30. Chambers, R.W. The chemistry of pseudouridine. IV. cyanoethylation. Biochemistry 4, 219–226 (1965).

    Article  CAS  Google Scholar 

  31. Chambers, R.W., Kurkov, V. & Shapiro, R. The chemistry of pseudouridine. synthesis of pseudouridine-5′-diphosphate. Biochemistry 2, 1192–1203 (1963).

    Article  CAS  Google Scholar 

  32. Yoshida, M. & Ukita, T. Modification of nucleosides and nucleotides. 8. The reaction rates of pseudouridine residues with acrylonitrile and its relation to the secondary structure of transfer ribonucleic acid. Biochim. Biophys. Acta 157, 466–475 (1968).

    Article  CAS  Google Scholar 

  33. Seeburg, P.H., Higuchi, M. & Sprengel, R. RNA editing of brain glutamate receptor channels: mechanism and physiology. Brain Res. Brain Res. Rev. 26, 217–229 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Kawahara, Y. et al. Frequency and fate of microRNA editing in human brain. Nucleic Acids Res. 36, 5270–5280 (2008).

    Article  CAS  Google Scholar 

  36. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  Google Scholar 

  37. Schaefer, M., Pollex, T., Hanna, K. & Lyko, F. RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 37, e12 (2009).

    Article  Google Scholar 

  38. Hartner, J.C. et al. Liver disintegration in the mouse embryo caused by deficiency in the RNA-editing enzyme ADAR1. J. Biol. Chem. 279, 4894–4902 (2004).

    Article  CAS  Google Scholar 

  39. Hartner, J.C., Walkley, C.R., Lu, J. & Orkin, S.H. ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon signaling. Nat. Immunol. 10, 109–115 (2009).

    Article  CAS  Google Scholar 

  40. George, C.X., Wagner, M.V. & Samuel, C.E. Expression of interferon-inducible RNA adenosine deaminase ADAR1 during pathogen infection and mouse embryo development involves tissue-selective promoter utilization and alternative splicing. J. Biol. Chem. 280, 15020–15028 (2005).

    Article  CAS  Google Scholar 

  41. Patterson, J.B. & Samuel, C.E. Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase. Mol. Cell. Biol. 15, 5376–5388 (1995).

    Article  CAS  Google Scholar 

  42. Ohlson, J., Enstero, M., Sjoberg, B.M. & Ohman, M. A method to find tissue-specific novel sites of selective adenosine deamination. Nucleic Acids Res. 33, e167 (2005).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Sakurai, M., Ohtsuki, T., Suzuki, T. & Watanabe, K. Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum. FEBS Lett. 579, 2767–2772 (2005).

    Article  CAS  Google Scholar 

  45. Suzuki, T., Suzuki, T., Wada, T., Saigo, K. & Watanabe, K. Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases. EMBO J. 21, 6581–6589 (2002).

    Article  CAS  Google Scholar 

  46. Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A. & Steinberg, S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 26, 148–153 (1998).

    Article  CAS  Google Scholar 

  47. Crooks, G.E., Hon, G., Chandonia, J.M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to the members of the Suzuki laboratory, especially to S. Okada and to H. Terajima for his experimental assistance and fruitful discussions on this study. Special thanks are due to Maze, Inc. for their support of computational work. 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.).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan

    Masayuki Sakurai, Takanori Yano, Hitomi Kawabata, Hiroki Ueda & Tsutomu Suzuki

Authors
  1. Masayuki Sakurai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takanori Yano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hitomi Kawabata
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroki Ueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tsutomu Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.S. developed and optimized the ICE method. T.Y. performed functional analyses of the identified sites. H.K. performed a genome-wide identification of the editing sites. H.U. carried out all computational work. T.S. designed and supervised all the work.

Corresponding author

Correspondence to Tsutomu Suzuki.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–12 and Supplementary Tables 1 & 2 (PDF 4147 kb)

Supplementary Tables

Supplementary Tables 3–6 (XLS 1317 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sakurai, M., Yano, T., Kawabata, H. et al. Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome. Nat Chem Biol 6, 733–740 (2010). https://doi.org/10.1038/nchembio.434

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.434

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing