Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2

Kelly J. Phelps, Kiet Tran, Tristan Eifler, Anna I. Erickson, Andrew J Fisher, Peter A. Beal

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Adenosine deaminases acting on RNA (ADARs) hydrolytically deaminate adenosines (A) in a wide variety of duplex RNAs and misregulation of editing is correlated with human disease. However, our understanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic domain. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interactions. Here we use a recently discovered RNA substrate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucleoside analog 8-azanebularine (8-azaN) into this RNA (and derived constructs) to mechanistically trap the protein-RNA complex without catalytic turnover for EMSA and ribonuclease footprinting analyses. EMSA showed that hADAR2-D requires duplex RNA and is sensitive to 2′-deoxy substitution at nucleotides opposite the editing site, the local sequence and 8-azaN nucleotide positioning on the duplex. Ribonuclease V1 footprinting shows that hADAR2-D protects ∼23 nt on the edited strand around the editing site in an asymmetric fashion (∼18 nt on the 5′ side and ∼5 nt on the 3′ side). These studies provide a deeper understanding of the ADAR catalytic domain-RNA interaction and new tools for biophysical analysis of ADAR-RNA complexes.

Original languageEnglish (US)
Pages (from-to)1123-1132
Number of pages10
JournalNucleic Acids Research
Volume43
Issue number2
DOIs
StatePublished - Jan 23 2015

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RNA Editing
RNA
Adenosine Deaminase
Enzymes
Catalytic Domain
Catalytic RNA
Ribonucleases
Nucleotides
Nucleosides
Adenosine

ASJC Scopus subject areas

  • Genetics

Cite this

Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. / Phelps, Kelly J.; Tran, Kiet; Eifler, Tristan; Erickson, Anna I.; Fisher, Andrew J; Beal, Peter A.

In: Nucleic Acids Research, Vol. 43, No. 2, 23.01.2015, p. 1123-1132.

Research output: Contribution to journalArticle

Phelps, Kelly J. ; Tran, Kiet ; Eifler, Tristan ; Erickson, Anna I. ; Fisher, Andrew J ; Beal, Peter A. / Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. In: Nucleic Acids Research. 2015 ; Vol. 43, No. 2. pp. 1123-1132.
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