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

Adenosine deaminases acting on RNA (ADARs) hy-drolytically deaminate adenosines (A) in a wide va-riety of duplex RNAs and misregulation of editing is correlated with human disease. However, our un-derstanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic do-main. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interac-tions. Here we use a recently discovered RNA sub-strate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucle-oside 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 op-posite 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