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

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.

An efficient system for selectively altering genetic information within mRNAs

“…They are similar to the rate constant given in one report of hADAR2 on the GluR-B R/G site (41). For all other hADAR2 substrates (30,31,40) they are about an order of magnitude slower. For reports on the hADAR2 deaminase domain on the highly editable Bdf2 R/G site (31), and for hADAR2 E488Q on the GluR-B R/G site (30), they are ∼2 orders of magnitude slower.…”

“…For all other hADAR2 substrates (30,31,40) they are about an order of magnitude slower. For reports on the hADAR2 deaminase domain on the highly editable Bdf2 R/G site (31), and for hADAR2 E488Q on the GluR-B R/G site (30), they are ∼2 orders of magnitude slower. We therefore conclude that our system edits with similar kinetics as WT ADARs on poorer substrates.…”

“…A previous study showed that a single mutation in a conserved loop near the catalytic center of WT hADAR2 (E488Q), increases the enzyme's catalytic rate (30), and it also increases the affinity, and activity, of the isolated deaminase domain for a substrate RNA (31). We added the E488Q mutation to both λN-DD and 4λN-DD in HEK293T cells (Figure 4A).…”

TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins

“…However the most prominent cause of false negatives is probably the strong preference of the ADAR catalytic domain for double-stranded RNA even without its dsRBDs (Macbeth et al, 2005, Eggington et al, 2011, Montiel-Gonzalez et al, 2013, Vogel et al, 2014, Vogel and Stafforst, 2014, Phelps et al, 2015). Although endogenous ADAR exhibits considerable plasticity, i.e., it edits regions of highly complex structure as well long stretches of duplex RNA, we assume that RBP-ADARcd proteins will not label their bound targets if they are composed exclusively of single stranded RNA.…”

Effects of Aicardi-Goutières syndrome mutations predicted from ADAR-RNA structures

“…28 Upon bonding in the ADAR active site, 8-azanebularine becomes hydrated mimicking the proposed intermediate thus forming a tight-binding dsRNA-ADAR complex amenable for structural studies. 29,30 The dsRNA-ADAR structure revealed approximately 20 bp of the dsRNA interacts with amino acid residues found entirely on loops between secondary structural elements of ADAR (Fig. 2).…”