How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs.
ABSTRACT: Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.
Project description:Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2?-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2?-deoxyadenosines in DNA/RNA hybrids.
Project description:Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.
Project description: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.
Project description:Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs in vivo remains largely unknown. Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein-protein interactions can guide substrate recognition for RNA editors.
Project description:The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA-binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.
Project description:Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system.
Project description:Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.
Project description:Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wild-type and ADAR mutant Caenorhabditis elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads and histone modifications associated with transcriptional silencing. Our data indicate that ADARs, through both direct and indirect mechanisms, are important for maintaining wild-type levels of many small RNAs in C. elegans.
Project description:OBJECTIVE: Hepatocellular carcinoma (HCC) is a heterogeneous tumour displaying a complex variety of genetic and epigenetic changes. In human cancers, aberrant post-transcriptional modifications, such as alternative splicing and RNA editing, may lead to tumour specific transcriptome diversity. DESIGN: By utilising large scale transcriptome sequencing of three paired HCC clinical specimens and their adjacent non-tumour (NT) tissue counterparts at depth, we discovered an average of 20 007 inferred A to I (adenosine to inosine) RNA editing events in transcripts. The roles of the double stranded RNA specific ADAR (Adenosine DeAminase that act on RNA) family members (ADARs) and the altered gene specific editing patterns were investigated in clinical specimens, cell models and mice. RESULTS: HCC displays a severely disrupted A to I RNA editing balance. ADAR1 and ADAR2 manipulate the A to I imbalance of HCC via their differential expression in HCC compared with NT liver tissues. Patients with ADAR1 overexpression and ADAR2 downregulation in tumours demonstrated an increased risk of liver cirrhosis and postoperative recurrence and had poor prognoses. Due to the differentially expressed ADAR1 and ADAR2 in tumours, the altered gene specific editing activities, which was reflected by the hyper-editing of FLNB (filamin B, β) and the hypo-editing of COPA (coatomer protein complex, subunit α), are closely associated with HCC pathogenesis. In vitro and in vivo functional assays prove that ADAR1 functions as an oncogene while ADAR2 has tumour suppressive ability in HCC. CONCLUSIONS: These findings highlight the fact that the differentially expressed ADARs in tumours, which are responsible for an A to I editing imbalance, has great prognostic value and diagnostic potential for HCC.
Project description:ADAR2 is a member of a family of RNA editing enzymes found in metazoa that bind double helical RNAs and deaminate select adenosines. We find that when human ADAR2 is overexpressed in the budding yeast Saccharomyces cerevisiae it substantially reduces the rate of cell growth. This effect is dependent on the deaminase activity of the enzyme, suggesting yeast transcripts are edited by ADAR2. Characterization of this novel set of RNA substrates provided a unique opportunity to gain insight into ADAR2's site selectivity. We used RNA-Seq. to identify transcripts present in S. cerevisiae subject to ADAR2-catalyzed editing. From this analysis, we identified 17 adenosines present in yeast RNAs that satisfied our criteria for candidate editing sites. Substrates identified include both coding and noncoding RNAs. Subsequent Sanger sequencing of RT-PCR products from yeast total RNA confirmed efficient editing at a subset of the candidate sites including BDF2 mRNA, RL28 intron RNA, HAC1 3'UTR RNA, 25S rRNA, U1 snRNA, and U2 snRNA. Two adenosines within the U1 snRNA sequence not identified as substrates during the original RNA-Seq. screen were shown to be deaminated by ADAR2 during the follow-up analysis. In addition, examination of the RNA sequence surrounding each edited adenosine in this novel group of ADAR2 sites revealed a previously unrecognized sequence preference. Remarkably, rapid deamination at one of these sites (BDF2 mRNA) does not require ADAR2's dsRNA-binding domains (dsRBDs). Human glioma-associated oncogene 1 (GLI1) mRNA is a known ADAR2 substrate with similar flanking sequence and secondary structure to the yeast BDF2 site discovered here. As observed with the BDF2 site, rapid deamination at the GLI1 site does not require ADAR2's dsRBDs.