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

ABSTRACT: 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.

SUBMITTER: Xu W

PROVIDER: S-EPMC5769745 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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

Xu Weijin W Rahman Reazur R Rosbash Michael M

RNA (New York, N.Y.) 20171110 2

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 identifie ...[more]

PMID: 29127211

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

Project description: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.

2017-08-19 | GSE102814 | GEO

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Project description: Not available

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