Biochemical Identification of A-to-I RNA Editing Sites by the Inosine Chemical Erasing (ICE) Method
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 wordsA-to-I RNA editing ADAR Inosine Cyanoethylation Acrylonitrile Reverse transcription PCR Direct sequencing Inosine chemical erasing (ICE)
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.).
- 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
- 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