Article

A method to identify RNA A-to-I editing targets using I-specific cleavage and exon array analysis

Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan
Molecular and Cellular Probes (Impact Factor: 1.85). 08/2012; 27(1). DOI: 10.1016/j.mcp.2012.08.008
Source: PubMed

ABSTRACT

RNA A-to-I editing is the most common single-base editing in the animal kingdom. Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Mouse knockout models deficient in ADAR activities show lethal phenotypes associated with defects in nervous system, failure of hematopoiesis and reduced tolerance to stress. While several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of ADARs remain unknown. Here we report a method to systematically analyze RNA A-to-I editing targets by combining I-specific cleavage and exon array analysis. Our results show that I-specific cleavage on editing sites causes more than twofold signal reductions in edited exons of known targets such as Gria2, Htr2c, Gabra3 and Cyfip2 in mice. This method provides an experimental approach for genome-wide analysis of RNA A-to-I editing targets with exon-level resolution. We believe this method will help expedite inquiry into the roles of RNA A-to-I editing in various biological processes and diseases.

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Available from: Hsueh-Wei Chang, Feb 25, 2014
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    • "These are based on the finding that glyoxal reacts with guanosine to form a stable adduct, whereas inosine glyoxal adducts are unstable. Guanosine glyoxal/borate adducts are resistant to RNase T1 digestion [50] [51] [52]. RNase T1 specifically cleaves RNA after guanosine or inosine, but is inhibited by guanosine glyoxal/borate adducts. "
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    • "These results, however, were not only restricted to the transcripts with probes on the microarray, but also limited due to the fact that a transcript's physical association with ADAR is not necessarily evidence of editing (Ohlson et al. 2005; Ohlson and Ohman 2007). More recently , Tseng et al. (2013) developed a method to detect RNA containing inosine by microarray and Sakurai and colleagues (Sakurai et al. 2010; Sakurai and Suzuki 2011) developed a protocol in which they used inosine cyanoethylation to block reverse transcription, which therefore allowed them to compare treated and untreated cDNAs to identify putative editing sites. While these protocols did not suffer the high falsepositive rates seen in other experiments, like the Ohlson protocol they required preexisting knowledge of the transcripts known (or suspected) to harbor editing sites, although it could be adapted to high-throughput approaches. "
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