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


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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    • "RNA editing can enhance the RNA and protein diversity [2]. Although five types of RNA editing have been discovered [3], the adenosine-to-inosine (A-to-I) editing is the most common type in higher eukaryotes [4–6]. The A-to-I editing may lead to changes in amino acid type and alternative splicing [7], thereby increasing the complexity of gene expression [8]. "
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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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    ABSTRACT: Adenosine to inosine (A > I) RNA editing, which is catalyzed by the ADAR family of proteins, is one of the fundamental mechanisms by which transcriptomic diversity is generated. Indeed, a number of genome-wide analyses have shown that A > I editing is not limited to a few mRNAs, as originally thought, but occurs widely across the transcriptome, especially in the brain. Importantly, there is increasing evidence that A > I editing is essential for animal development and nervous system function. To more efficiently characterize the complete catalog of ADAR events in the mammalian transcriptome we developed a high-throughput protocol to identify A > I editing sites, which exploits the capacity of glyoxal to protect guanosine, but not inosine, from RNAse T1 treatment, thus facilitating extraction of RNA fragments with inosine bases at their termini for high-throughput sequencing. Using this method we identified 665 editing sites in mouse brain RNA, including most known sites and suite of novel sites that include nonsynonymous changes to protein-coding genes, hyperediting of genes known to regulate p53, and alterations to non-protein-coding RNAs. This method is applicable to any biological system for the de novo discovery of A > I editing sites, and avoids the complicated informatic and practical issues associated with editing site identification using traditional RNA sequencing data. This approach has the potential to substantially increase our understanding of the extent and function of RNA editing, and thereby to shed light on the role of transcriptional plasticity in evolution, development, and cognition.
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