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.     

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.     

Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4⁺ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1.     

Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase.

Double-stranded RNA adenosine deaminase (dsRAD), previously called the double-stranded RNA (dsRNA) unwinding/modifying activity, modifies adenosines to inosines within dsRNA. We used ribonuclease U2 and a mutant of ribonuclease T1 to map the sites of modification in several RNA duplexes. We found that dsRAD had a 5' neighbor preference (A = U > C > G) but no apparent 3' neighbor preference. Further, the proximity of the strand termini affected whether an adenosine was modified. Most importantly, dsRAD exhibited selectivity, modifying a minimal number of adenosines in short dsRNAs. Our results suggest that the specific editing of glutamate receptor subunit B mRNA could be performed in vivo by dsRAD without the aid of specificity factors, and support the hypothesis that dsRAD is responsible for hypermutations in certain RNA viruses.     

Modulation of ADAR1 editing activity by Z-RNA in vitro

RNA editing by A-to-I modification has been recognized as an important molecular mechanism for generating RNA and protein diversity. In mammals, it is mediated by a family of adenosine deaminases that act on RNAs (ADARs). The large version of the editing enzyme ADAR1 (ADAR1-L), expressed from an interferon-responsible promoter, has a Z-DNA/Z-RNA binding domain at its N-terminus. We have tested the in vitro ability of the enzyme to act on a 50 bp segment of dsRNA with or without a Z-RNA forming nucleotide sequence. A-to-I editing efficiency is markedly enhanced in presence of the sequence favoring Z-RNA. In addition, an alteration in the pattern of modification along the RNA duplex becomes evident as reaction times decrease. These results suggest that the local conformation of dsRNA molecules might be an important feature for target selectivity by ADAR1 and other proteins with Z-RNA binding domains.     

Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNA-mediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.     

ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to inosines in double-stranded RNAs including miRNA precursors. A to I editing is widespread and required for normal life. By comparing deep sequencing data of brain miRNAs from wild-type and ADAR2 deficient mouse strains, we detect editing sites and altered miRNA processing at high sensitivity. We detect 48 novel editing events in miRNAs. Some editing events reach frequencies of up to 80%. About half of all editing events depend on ADAR2 while some miRNAs are preferentially edited by ADAR1. Sixty-four percent of all editing events are located within the seed region of mature miRNAs. For the highly edited miR-3099, we experimentally prove retargeting of the edited miRNA to novel 3′ UTRs. We show further that an abundant editing event in miR-497 promotes processing by Drosha of the corresponding pri-miRNA. We also detect reproducible changes in the abundance of specific miRNAs in ADAR2-deficient mice that occur independent of adjacent A to I editing events. This indicates that ADAR2 binding but not editing of miRNA precursors may influence their processing. Correlating with changes in miRNA abundance we find misregulation of putative targets of these miRNAs in the presence or absence of ADAR2.     

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.     

The role of RNA editing by ADARs in RNAi

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). Here we demonstrate that ADARs are not required for RNA interference (RNAi) and that they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. We find, however, that transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals we detect edited dsRNA corresponding to transgenes, and we propose that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals.     

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.     

Host response to polyomavirus infection is modulated by RNA adenosine deaminase ADAR1 but not by ADAR2

Adenosine deaminases acting on RNA (ADARs) catalyze the C-6 deamination of adenosine (A) to produce inosine (I), which behaves as guanine (G), thereby altering base pairing in RNAs with double-stranded character. Two genes, adar1 and adar2, are known to encode enzymatically active ADARs in mammalian cells. Furthermore, two size forms of ADAR1 are expressed by alternative promoter usage, a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus. ADAR2 is also a constitutively expressed nuclear protein. Extensive A-to-G substitution has been described in mouse polyomavirus (PyV) RNA isolated late times after infection, suggesting modification by ADAR. To test the role of ADAR in PyV infection, we used genetically null mouse embryo fibroblast cells deficient in either ADAR1 or ADAR2. The single-cycle yields and growth kinetics of PyV were comparable between adar1(-/-) and adar2(-/-) genetic null fibroblast cells. While large T antigen was expressed to higher levels in adar1(-/-) cells than adar2(-/-) cells, less difference was seen in VP1 protein expression levels between the two knockout MEFs. However, virus-induced cell killing was greatly enhanced in PyV-infected adar1(-/-) cells compared to that of adar2(-/-) cells. Complementation with p110 protected cells from PyV-induced cytotoxicity. UV-irradiated PyV did not display any enhanced cytopathic effect in adar1(-/-) cells. Reovirus and vesicular stomatitis virus single-cycle yields were comparable between adar1(-/-) and adar2(-/-) cells, and neither reovirus nor VSV showed enhanced cytotoxicity in adar1(-/-)-infected cells. These results suggest that ADAR1 plays a virus-selective role in the host response to infection.     

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.