Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to produce inosines within RNAs that are largely double-stranded (ds). Like most dsRNA binding proteins, the enzymes will bind to any dsRNA without apparent sequence specificity. However, once bound, ADARs deaminate certain adenosines more efficiently than others. Most of what is known about the intrinsic deamination specificity of ADARs derives from analyses of Xenopus ADAR1. In addition to ADAR1, mammalian cells have a second ADAR, named ADAR2; the deamination specificity of this enzyme has not been rigorously studied. Here we directly compare the specificity of human ADAR1 and ADAR2. We find that, like ADAR1, ADAR2 has a 5' neighbor preference (A approximately U > C = G), but, unlike ADAR1, also has a 3' neighbor preference (U = G > C = A). Simultaneous analysis of both neighbor preferences reveals that ADAR2 prefers certain trinucleotide sequences (UAU, AAG, UAG, AAU). In addition to characterizing ADAR2 preferences, we analyzed the fraction of adenosines deaminated in a given RNA at complete reaction, or the enzyme's selectivity. We find that ADAR1 and ADAR2 deaminate a given RNA with the same selectivity, and this appears to be dictated by features of the RNA substrate. Finally, we observed that Xenopus and human ADAR1 deaminate the same adenosines on all RNAs tested, emphasizing the similarity of ADAR1 in these two species. Our data add substantially to the understanding of ADAR2 specificity, and aid in efforts to predict which ADAR deaminates a given editing site adenosine in vivo.     

Analysis of the RNA-editing reaction of ADAR2 with structural and fluorescent analogues of the GluR-B R/G editing site

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.     

ADAR2 A-->I editing: site selectivity and editing efficiency are separate events

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A→I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative effect on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.     

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

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.     

Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2

Hepatitis delta virus (HDV) is a subviral human pathogen that uses specific RNA editing activity of the host to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the longer form (HDAg-L), which is required for RNA packaging but which is a potent trans-dominant inhibitor of HDV RNA replication. Editing in infected cells is thought to be catalyzed by one or more of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). We examined the effects of increased ADAR1 and ADAR2 expression on HDV RNA editing and replication in transfected Huh7 cells. We found that both ADARs dramatically increased RNA editing, which was correlated with strong inhibition of HDV RNA replication. While increased HDAg-L production was the primary mechanism of inhibition, we observed at least two additional means by which ADARs can suppress HDV replication. High-level expression of both ADAR1 and ADAR2 led to extensive hyperediting at non-amber/W sites and subsequent production of HDAg variants that acted as trans-dominant inhibitors of HDV RNA replication. Moreover, we also observed weak inhibition of HDV RNA replication by mutated forms of ADARs defective for deaminase activity. Our results indicate that HDV requires highly regulated and selective editing and that the level of ADAR expression can play an important role: overexpression of ADARs inhibits HDV RNA replication and compromises virus viability.     

Modulation of microRNA processing and expression through RNA editing by ADAR deaminases

Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri-miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri-miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.     

Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases

Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.     

Conformational changes that occur during an RNA-editing adenosine deamination reaction

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.     

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.     

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.