Other Expression profiling by high throughput sequencing
Summary
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
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