Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein

• DOI: 10.1261/rna.064691.117
• Publication Date: 2017-11-11T01:25:18Z
• Authors: Weijin Xu; Reazur Rahman; Michael Rosbash
• Keywords: 0303 health sciences; Adenosine; Binding Sites; Adenosine Deaminase; Sequence Analysis, RNA; Recombinant Fusion Proteins; RNA-Binding Proteins; Article; Heterogeneous-Nuclear Ribonucleoproteins; Inosine; 03 medical and health sciences; Catalytic Domain; Mutation; Animals; Drosophila Proteins; RNA Editing

We previously developed TRIBE, a method for the identification of cell-specific RNA-binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA-editing enzyme ADAR and performs adenosine-to-inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing 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, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.