Structure and Specific RNA Binding of ADAR2 Double-Stranded RNA Binding Motifs

Abstract

Adenosine deaminases that act on RNA (ADARs) site-selectively modify adenosines to inosines within RNA transcripts, thereby recoding genomic information. How ADARs select specific adenosine moieties for deamination is poorly understood. Here, we report NMR structures of the two double-stranded RNA binding motifs (dsRBMs) of rat ADAR2 and an NMR chemical shift perturbation study of the interaction of the two dsRBMs with a 71 nucleotide RNA encoding the R/G site of the GluR-B. We have identified the protein and the RNA surfaces involved in complex formation, allowing us to present an NMR-based model of the complex. We have found that dsRBM1 recognizes a conserved pentaloop, whereas dsRBM2 recognizes two bulged bases adjacent to the editing site, demonstrating RNA structure-dependent recognition by the ADAR2 dsRBMs. In vitro mutagenesis studies with both the protein and the RNA further support our structural findings.

Editor meets silencer: crosstalk between RNA editing and RNA interference

“…3), indicating a significant difference in their RNA-substrate interactions, possibly through their dsRBDs (different numbers and spacing between different dsRBDs) 53 . The distinctive site selectivity of ADAR1 and ADAR2 could also be mediated through functional interactions between the two monomers of ADAR1 or ADAR2, as such interactions possibly position specific adenosine residues relative to the catalytic centre of ADAR 53,54 .…”

Substrate-dependent Contribution of Double-stranded RNA-binding Motifs to ADAR2 Function

“…The contribution of dsRBM1 to the site-selective editing of ADAR2 seems to be substrate dependent, whereas dsRBM2 is critical for enzymatic activity with all substrates examined ( Figure 5), despite the high degree of conservation at both the levels of amino acid sequence and protein structure (Tian et al, 2004;Stefl et al, 2006). This functional inequality between dsRBM1 and dsRBM2 could result from subtle sequence or structural differences affecting RNA-protein interactions or the relative position of each motif in the ADAR2 protein.…”

“…The most extensively studied substrates of Ato-I conversion are transcripts encoding ionotropic glutamate receptor (GluR) subunits and the 2C-subtype of the serotonin receptor (5-HT 2C R), in which editing leads to nonsynonymous codon changes that generate channels with altered electrophysiologic and ion permeation properties (Seeburg and Hartner, 2003) and receptors with decreased G protein-coupling efficiency (Burns et al, 1997;Niswender et al, 1999;Berg et al, 2001). A-to-I modifications have also been described in nontranslated RNAs and noncoding regions of mRNA transcripts, suggesting that such RNA modifications affect other aspects of RNA function, including splicing, translation efficiency, nuclear retention, and transcript stability (Rueter et al, 1999;Athanasiadis et al, 2004;Blow et al, 2004;Kim et al, 2004;Levanon et al, 2004;DeCerbo and Carmichael, 2005;Prasanth et al, 2005).ADAR2 displays a modular organization with two tandem dsRNA-binding motifs (dsRBMs) connected by a flexible linker and a conserved adenosine deaminase domain toward the carboxy terminus, for which the structures have recently been determined (Macbeth et al, 2005;Stefl et al, 2006). The dsRBM is a highly conserved 65-75 amino acid (aa) domain, present in many eukaryotic proteins with diverse cellular functions, that forms an ␣1-␤1-␤2-␤3-␣2 topology in which the two ␣-helices are packed along a face of a three-stranded antiparallel ␤-sheet, with most of the potential RNA-binding residues exposed on one surface (FierroMonti and Mathews, 2000;Tian et al, 2004).…”

“…Structural analyses of dsRBMs complexed to short, synthetic RNA duplexes have suggested that dsRBM binding is independent of RNA sequence, because the majority of dsRBM-RNA interactions involve direct contact with the 2Ј-hydroxyl groups of the ribose sugars and direct or water-mediated contacts with nonbridging oxygen residues of the phosphodiester backbone (Ryter and Schultz, 1998;Ramos et al, 2000;Blaszczyk et al, 2004). The two dsRBMs of ADAR2, with Ͼ80% amino acid sequence similarity, adopt the same fold as all other members of the dsRBM family, although the two domains differ slightly from one another in the orientation of ␣1 helix relative to the other secondary structural elements (Stefl et al, 2006). Like other dsRBM-containing proteins, ADAR2 demonstrates a high-affinity for dsRNA and can edit up to 50% of the adenosine moieties in perfect dsRNA Liu et al, 1999;Lehmann and Bass, 2000;Cho et al, 2003;Dawson et al, 2004).…”

“…The relative nucleolar fluorescence for the ⌬dsRBM1 mutant was significantly less than for the fusion protein lacking dsRBM2 (p Ͻ 0.02), suggesting a greater role for dsRBM1 in the subnuclear compartmentalization of ADAR2. Independent substitutions of K 127 and K 281 decreased relative nucleolar fluorescence to the same extent (K127A and K281A); however, the magnitude of this effect was less than that observed for the dsRBM deletions ( Figure 2), suggesting that additional contacts between the dsRBMs and nucleolar binding site(s) are required for normal nucleolar localization.Because deletion or even subtle point mutations of ADAR2 dsRBMs could result in structural alterations that cause ADAR2 mislocalization, we also examined the subcellular localization of eYFP when expressed as a fusion protein with either dsRBM1 (aa 74 -147) or dsRBM2 (aa 231-301) alone ( Figure 2C); the precise borders for each motif were based upon amino acid sequence homology to other dsRBMcontaining proteins and the recently resolved NMR structure for these domains in ADAR2 (Fierro-Monti and Mathews, 2000;Stefl et al, 2006). Both eYFP-dsRBM1 and eYFP-dsRBM2 were localized to the cytoplasm and nucleus, presumably because of the absence of a nuclear localization signal and the passive diffusion of in these small fusion proteins (ϳ40 kDa) through the nuclear pore (Suntharalingam and Wente, 2003); however, eYFP-dsRBM1 was localized to nucleoli significantly better than eYFP-dsRBM2 (p Ͻ 0.001; Figure 2C), further confirming a nonequivalent role for these highly …”