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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

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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.

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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.

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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.).

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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

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  1. Masayuki Sakurai
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  2. Takanori Yano
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  3. Hitomi Kawabata
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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.

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Correspondence to Tsutomu Suzuki.

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The authors declare no competing financial interests.

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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

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