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.     

Regulation of glutamate receptor B pre-mRNA splicing by RNA editing

RNA-editing enzymes of the ADAR family convert adenosines to inosines in double-stranded RNA substrates. Frequently, editing sites are defined by base-pairing of the editing site with a complementary intronic region. The glutamate receptor subunit B (GluR-B) pre-mRNA harbors two such exonic editing sites termed Q/R and R/G. Data from ADAR knockout mice and in vitro editing assays suggest an intimate connection between editing and splicing of GluR-B pre-mRNA.

By comparing the events at the Q/R and R/G sites, we can show that editing can both stimulate and repress splicing efficiency. The edited nucleotide, but not ADAR binding itself, is sufficient to exert this effect. The presence of an edited nucleotide at the R/G site reduces splicing efficiency of the adjacent intron facilitating alternative splicing events occurring downstream of the R/G site.

Lack of editing inhibits splicing at the Q/R site. Editing of both the Q/R nucleotide and an intronic editing hotspot are required to allow efficient splicing. Inefficient intron removal may ensure that only properly edited mRNAs become spliced and exported to the cytoplasm.

     

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.     

Editing of AMPA and Serotonin 2C Receptors in Individual Central Neurons,Controlling Wakefulness

(1) Pre-mRNA editing of serotonin 2C (5-HT_2C) and glutamate (Glu) receptors (R) influences higher brain functions and pathological states such as epilepsy, amyotrophic lateral sclerosis, and depression. Adenosine deaminases acting on RNA (ADAR1–3) convert adenosine to inosine on synthetic RNAs, analogous to 5-HT_2cR and GluR. The order of editing as well as mechanisms controlling editing in native neurons is unknown. (2) With single-cell RT-PCR we investigated the co-expression of ADAR genes with GluR and 5-HT_2CR and determined the editing status at known sites in the hypothalamic tuberomamillary nucleus, a major center for wakefulness and arousal. (3) The most frequently expressed enzymes were ADAR1, followed by ADAR2. The Q/R site of GluR2 was always fully edited. Editing at the R/G site in the GluR2 (but not GluR4) subunit was co-ordinated with ADAR expression: maximal editing was found in neurons expressing both ADAR2 splice variants of the deaminase domain and lacking ADAR3. (4) Editing of the 5-HT_2CR did not correlate with ADAR expression. The 5-HT_2CR mRNA was always edited at A, in the majority of cells at B sites and variably edited at E, C and D sites. A negative correlation was found between editing of C and D sites. The GluR4 R/G site editing was homogeneous within individuals: it was fully edited in all neurons obtained from 12 rats and under-edited in six neurons obtained from three rats. (5) We conclude that GluR2 R/G editing is controlled at the level of ADAR2 and therefore this enzyme may be a target for pharmacotherapy. On the other hand, further factors/enzymes besides ADAR must control or influence 5-HT_2CR and GluR pre-mRNA editing in native neurons; our data indicate that these factors vary between individuals and could be predictors of psychiatric disease.     

ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia

ADAR2 is a nuclear enzyme essential for GluR2 pre-mRNA editing at Q/R site-607, which gates Ca2+ entry through AMPA receptor channels. Here, we show that forebrain ischemia in adult rats selectively reduces expression of ADAR2 enzyme and, hence, disrupts RNA Q/R site editing of GluR2 subunit in vulnerable neurons. Recovery of GluR2 Q/R site editing by expression of exogenous ADAR2b gene or a constitutively active CREB, VP16-CREB, which induces expression of endogenous ADAR2, protects vulnerable neurons in the rat hippocampus from forebrain ischemic insult. Generation of a stable ADAR2 gene silencing by delivering small interfering RNA (siRNA) inhibits GluR2 Q/R site editing, leading to degeneration of ischemia-insensitive neurons. Direct introduction of the Q/R site edited GluR2 gene, GluR2(R607), rescues ADAR2 degeneration. Thus, ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons in the rat hippocampus to forebrain ischemia.     

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.     

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.     

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.     

A method to find tissue-specific novel sites of selective adenosine deamination

Site-selective adenosine (A) to inosine (I) RNA editing by the ADAR enzymes has been found in a variety of metazoan from fly to human. Here we describe a method to detect novel site-selective A to I editing that can be used on various tissues as well as species. We have shown previously that there is a preference for ADAR2-binding to selectively edited sites over non-specific interactions with random sequences of double-stranded RNA. The method utilizes immunoprecipitation (IP) of intrinsic RNA–protein complexes to extract substrates subjected to site-selective editing in vivo, in combination with microarray analyses of the captured RNAs. We show that known single sites of A to I editing can be detected after IP using an antibody against the ADAR2 protein. The RNA substrates were verified by RT–PCR, RNase protection and microarray. Using this method it is possible to uniquely identify novel single sites of selective A to I editing.     

Induction of protein translation by ADAR1 within living cell nuclei is not dependent on RNA editing

Translation of mRNA is usually cytoplasmic. We report that the RNA editing enzyme ADAR1, which catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates, induces translation within the nucleus, possibly at the surface of the nucleolus. This activity does not depend on RNA editing. Two regions within ADAR1 are defined that act independently of each other to induce translation: the first includes the double-stranded RNA binding domains (DRBMs) of ADAR1 while the second maps to the C-terminal portion of the catalytic domain. Point mutations within each domain are identified that reduce nuclear translation; those in the DRBM region are also known to diminish RNA binding. This report adds to the growing functionality ascribed to the nucleus.     

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.