Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein. Drosophila melanogaster

Most current methods to identify cell-specific RNA binding protein (RBP) targets require analyzing an extract, a strategy that is problematic with small amounts of material. We previously addressed this issue by developing TRIBE, a method that expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR. TRIBE performs Adenosine-to-Inosine editing on candidate RNA targets of the RBP. However, target identification is limited by the efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites than TRIBE, many of which are also edited by TRIBE but at a much lower editing frequency. The data have mechanistic implications for the enhanced editing activity of the HyperADARcd as part of a RBP fusion protein and also indicate that HyperTRIBE more faithfully recapitulates the known binding specificity of its RBP than TRIBE. Overall design: E488Q mutation has been reported to increase ADAR editing efficiency. We adopted this mutation in TRIBE technique to address its false negative problem