Biochemical and Transcriptome-Wide Identification of A-to-I RNA Editing Sites by ICE-Seq

Abstract

Inosine (I) is a modified adenosine (A) in RNA. In Metazoa, I is generated by hydrolytic deamination of A, catalyzed by adenosine deaminase acting RNA (ADAR) in a process called A-to-I RNA editing. A-to-I RNA editing affects various biological processes by modulating gene expression. In addition, dysregulation of A-to-I RNA editing results in pathological consequences. I on RNA strands is converted to guanosine (G) during cDNA synthesis by reverse transcription. Thus, the conventional method used to identify A-to-I RNA editing sites compares cDNA sequences with their corresponding genomic sequences. Combined with deep sequencing, this method has been applied to transcriptome-wide screening of A-to-I RNA editing sites. This approach, however, produces a large number of false positives mainly owing to mapping errors. To address this issue, we developed a biochemical method called inosine chemical erasing (ICE) to reliably identify genuine A-to-I RNA editing sites. In addition, we applied the ICE method combined with RNA-seq, referred to as ICE-seq, to identify transcriptome-wide A-to-I RNA editing sites. In this chapter, we describe the detailed protocol for ICE-seq, which can be applied to various sources and taxa.

Keywords: A-to-I RNA editing; ADAR; ICE method; ICE-seq; Inosine; RDD.