In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.     

Molecular dynamics simulations and free energy calculations of base flipping in dsRNA

The family of adenosine deaminases acting on RNA (ADARs) targets adenosines in RNA that is mainly double stranded. Some substrates are promiscuously deaminated whereas others, such as the mammalian glutamate receptor B (gluR-B) pre-mRNA, are more selectively deaminated. Many DNA/RNA-base modification enzymes use a base flipping mechanism to be able to reach their target base and it is believed that ADARs function in a similar way. In this study we used molecular dynamics (MD) simulations to describe two sites on the gluR-B pre-mRNA, the selectively targeted R/G site and the nontargeted 46 site, in an attempt to explain the substrate specificity. We used regular MD and also a forced base flipping method with umbrella sampling to calculate the free energy of base opening. Spontaneous opening of the mismatched adenosine was observed for the R/G site but not for the 46 site.     

A structural determinant required for RNA editing

RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.     

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.     

ADARs: allies or enemies? The importance of A‐to‐I RNA editing in human disease: from cancer to HIV‐1

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine (A) to inosine (I) in nuclear‐encoded RNAs and viral RNAs. The activity of ADARs has been demonstrated to be essential in mammals and serves to fine‐tune different proteins and modulate many molecular pathways. Recent findings have shown that ADAR activity is altered in many pathological tissues. Moreover, it has been shown that modulation of RNA editing is important for cell proliferation and migration, and has a protective effect on ischaemic insults. This review summarises available recent knowledge on A‐to‐I RNA editing and ADAR enzymes, with particular attention given to the emerging role played by these enzymes in cancer, some infectious diseases and immune‐mediated disorders.     

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.     

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.     

Pentatricopeptide repeat proteins constrain genome evolution in chloroplasts

Higher plants encode hundreds of pentatricopeptide repeat proteins (PPRs) that are involved in several types of RNA processing reactions. Most PPR genes are predicted to be targeted to chloroplasts or mitochondria, and many are known to affect organellar gene expression. In some cases, RNA binding has been directly demonstrated, and the sequences of the cis-elements are known. In this work, we demonstrate that RNA cis-elements recognized by PPRs are constrained in chloroplast genome evolution. Cis-elements for two PPR genes and several RNA editing sites were analyzed for sequence changes by pairwise nucleotide substitution frequency, pairwise indel frequency, and maximum likelihood (ML) phylogenetic distances. All three of these analyses demonstrated that sequences within the cis-element are highly conserved compared with surrounding sequences. In addition, we have compared sequences around chloroplast editing sites and homologous sequences in species that lack an editing site due to the presence of a genomic T. Cis-elements for RNA editing sites are highly conserved in angiosperms; by contrast, comparable sequences around a genomically encoded T exhibit higher rates of nucleotide substitution, higher frequencies of indels, and greater ML distances. The loss in requirement for editing to create the ndhD start codon has resulted in the conversion of the PPR gene responsible for editing that site to a pseudogene. We show that organellar dependence on nuclear-encoded PPR proteins for gene expression has constrained the evolution of cis-elements that are required at the level of RNA processing. Thus, the expansion of the PPR gene family in plants has had a dramatic effect on the evolution of plant organelle genomes.     

Identification of widespread ultra-edited human RNAs

Adenosine-to-inosine modification of RNA molecules (A-to-I RNA editing) is an important mechanism that increases transciptome diversity. It occurs when a genomically encoded adenosine (A) is converted to an inosine (I) by ADAR proteins. Sequencing reactions read inosine as guanosine (G); therefore, current methods to detect A-to-I editing sites align RNA sequences to their corresponding DNA regions and identify A-to-G mismatches. However, such methods perform poorly on RNAs that underwent extensive editing ("ultra"-editing), as the large number of mismatches obscures the genomic origin of these RNAs. Therefore, only a few anecdotal ultra-edited RNAs have been discovered so far. Here we introduce and apply a novel computational method to identify ultra-edited RNAs. We detected 760 ESTs containing 15,646 editing sites (more than 20 sites per EST, on average), of which 13,668 are novel. Ultra-edited RNAs exhibit the known sequence motif of ADARs and tend to localize in sense strand Alu elements. Compared to sites of mild editing, ultra-editing occurs primarily in Alu-rich regions, where potential base pairing with neighboring, inverted Alus creates particularly long double-stranded RNA structures. Ultra-editing sites are underrepresented in old Alu subfamilies, tend to be non-conserved, and avoid exons, suggesting that ultra-editing is usually deleterious. A possible biological function of ultra-editing could be mediated by non-canonical splicing and cleavage of the RNA near the editing sites.     

Editing independent effects of ADARs on the miRNA/siRNA pathways

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.     

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.     

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.