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Biochemical Identification of A-to-I RNA Editing Sites by the Inosine Chemical Erasing (ICE) Method

  • Masayuki Sakurai
  • Tsutomu SuzukiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 718)

Abstract

Adenosine-to-inosine (A-to-I) RNA editing is a biologically important posttranscriptional processing event involved in the transcriptome diversification. The most conventional method of editing site identification is to compare the cDNA sequence with its corresponding genomic sequence; however, using this method, it is difficult to discriminate between guanosine residue that originated from inosine and errors or noise in the sequencing chromatograms. To address this issue, we developed the inosine chemical erasing (ICE) method to identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription PCR. Since this method requires only a limited quantity of total RNA, it can be used in the genome-wide profiling of A-to-I editing sites in tissues and cells from various organisms, including clinical specimens.

Key words

A-to-I RNA editing ADAR Inosine Cyanoethylation Acrylonitrile Reverse transcription PCR Direct sequencing Inosine chemical erasing (ICE) 

Notes

Acknowledgments

We are grateful to the Suzuki lab members, including Takanori Yano, Hiroki Ueda, Hitomi Kawabata, and Shunpei Okada, for their computational and experimental assistance and fruitful discussions on this study. 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 (NEDO) (to T. S.).

References

  1. 1.
    Bass, B. L. (2002) RNA editing by adenosine deaminases that act on RNA. Annu Rev Biochem 71, 817–846.PubMedCrossRefGoogle Scholar
  2. 2.
    Higuchi, M., Maas, S., Single, F. N., Hartner, J., Rozov, A., Burnashev, N., Feldmeyer, D., Sprengel, R., and Seeburg, P. H. (2000) Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406, 78–81.PubMedCrossRefGoogle Scholar
  3. 3.
    Wang, Q., Khillan, J., Gadue, P., and Nishikura, K. (2000) Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 290, 1765–1768.PubMedCrossRefGoogle Scholar
  4. 4.
    Wang, Q., Miyakoda, M., Yang, W., Khillan, J., Stachura, D. L., Weiss, M. J., and Nishikura, K. (2004) Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. J Biol Chem 279, 4952–4961.PubMedCrossRefGoogle Scholar
  5. 5.
    Jepson, J. E., and Reenan, R. A. (2008) RNA editing in regulating gene expression in the brain. Biochim Biophys Acta 1779, 459–470.PubMedGoogle Scholar
  6. 6.
    Nishikura, K. (2006) Editor meets silencer: crosstalk between RNA editing and RNA interference. Nat Rev Mol Cell Biol 7, 919–931.PubMedCrossRefGoogle Scholar
  7. 7.
    Maas, S., Kawahara, Y., Tamburro, K. M., and Nishikura, K. (2006) A-to-I RNA editing and human disease. RNA Biol 3, 1–9.PubMedGoogle Scholar
  8. 8.
    Paz, N., Levanon, E. Y., Amariglio, N., Heimberger, A. B., Ram, Z., Constantini, S., Barbash, Z. S., Adamsky, K., Safran, M., Hirschberg, A., Krupsky, M., Ben-Dov, I., Cazacu, S., Mikkelsen, T., Brodie, C., Eisenberg, E., and Rechavi, G. (2007) Altered adenosine-to-inosine RNA editing in human cancer. Genome Res 17, 1586–1595.PubMedCrossRefGoogle Scholar
  9. 9.
    Burns, C. M., Chu, H., Rueter, S. M., Hutchinson, L. K., Canton, H., Sanders-Bush, E., and Emeson, R. B. (1997) Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387, 303–308.PubMedCrossRefGoogle Scholar
  10. 10.
    Nishimoto, Y., Yamashita, T., Hideyama, T., Tsuji, S., Suzuki, N., and Kwak, S. (2008) Determination of editors at the novel A-to-I editing positions. Neurosci Res 61, 201–206.PubMedCrossRefGoogle Scholar
  11. 11.
    Sakurai, M., Yano, T., Kawabata, H., Ueda, H., and Suzuki, T. (2010) Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome. Nat Chem Biol 6, 733–740.Google Scholar
  12. 12.
    Yoshida, M. and Ukita, T. (1968) Modification of nucleosides and nucleotides. VII. Selective cyanoethylation of inosine and pseudouridine in yeast transfer ribonucleic acid. Biochim Biophys Acta 157, 455–465.PubMedGoogle Scholar
  13. 13.
    Hoopengardner, B., Bhalla, T., Staber, C., and Reenan, R. (2003) Nervous system targets of RNA editing identified by comparative genomics. Science 301, 832–836.PubMedCrossRefGoogle Scholar
  14. 14.
    Sommer, B., Kohler, M., Sprengel, R., and Seeburg, P. H. (1991) RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67, 11–19.PubMedCrossRefGoogle Scholar
  15. 15.
    Sato, K., Hamada, M., Asai, K., and Mituyama, T. (2009) CENTROIDFOLD: a web server for RNA secondary structure prediction. Nucleic Acids Res 37, W277–W280.PubMedCrossRefGoogle Scholar
  16. 16.
    Hamada, M., Kiryu, H., Sato, K., Mituyama, T., and Asai, K. (2009) Prediction of RNA secondary structure using generalized centroid estimators. Bioinformatics 25, 465–473.PubMedCrossRefGoogle Scholar
  17. 17.
    Washietl, S., Hofacker, I. L., and Stadler, P. F. (2005) Fast and reliable prediction of noncoding RNAs. Proc Natl Acad Sci U S A 102, 2454–2459.PubMedCrossRefGoogle Scholar
  18. 18.
    Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Chemistry and Biotechnology, Graduate School of EngineeringUniversity of TokyoBunkyo-kuJapan

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