Detection of inosine in messenger RNA by inosine-specific cleavage

Double-stranded RNA adenosine deaminases catalyze the conversion of adenosine to inosine within double-stranded RNA. A few candidate biological substrates for these enzymes have been discovered by noticing discrepancies between genomic and cDNA sequences. Toward the goal of finding a systematic approach to identify new deaminase substrates, we developed a method to cleave RNA specifically after inosine and an amplification strategy to identify the cleavage sites. We tested our method on a candidate substrate, the messenger RNA for glutamate receptor subunit B (GluR-B). We detected cleavage of the endogenous GluR-B message from rat brain at two known RNA editing sites, thus providing the first direct evidence for the presence of inosine at these sites. The described method will facilitate the mapping of inosines within RNA and, most importantly, will provide a way to identify new deaminase substrates.