DETECTION OF INOSINE IN MESSENGER-RNA BY INOSINE-SPECIFIC CLEAVAGE

Double-stranded RNA adenosine deaminases catalyze the conversion of ad enosine to inosine within double-stranded RNA. A few candidate biologi cal substrates for these enzymes have been discovered by noticing disc repancies between genomic and cDNA sequences. Toward the goal of findi ng a systematic approach to identify new deaminase substrates, mie dev eloped a method to cleave RNA specifically after inosine and an amplif ication strategy to identify the cleavage sites, We tested our method on a candidate substrate, the messenger RNA for glutamate receptor sub unit B (GluR-B), We detected cleavage of the endogenous GluR-B message from rat brain at two known RNA editing sites, thus providing the fir st direct evidence for the presence of inosine at these sites, The des cribed method will facilitate the mapping of inosines within RNA and, most importantly, will provide a way to identify new deaminase substra tes.