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

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 ident...

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Bibliographic Details
Published in:RNA (Cambridge) 2018-02, Vol.24 (2), p.173-182
Main Authors: Xu, Weijin, Rahman, Reazur, Rosbash, Michael
Format: Article
Language:English
Subjects:
Adenosine
Adenosine - metabolism
Adenosine Deaminase - genetics
Animals
Binding Sites
Catalytic Domain
Drosophila Proteins - genetics
Drosophila Proteins - metabolism
Editing
Heterogeneous-Nuclear Ribonucleoproteins - genetics
Heterogeneous-Nuclear Ribonucleoproteins - metabolism
Inosine - metabolism
Mutation
Recombinant Fusion Proteins - metabolism
Ribonucleic acid
RNA
RNA Editing
RNA-binding protein
RNA-Binding Proteins - metabolism
Sequence Analysis, RNA
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Summary: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.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.064691.117