Inositol Hexakisphosphate Is Bound in the ADAR2 Core and Required for RNA Editing (original) (raw)
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An RNA Editor, Adenosine Deaminase Acting on Double-Stranded RNA (ADAR1)
Journal of Interferon & Cytokine Research, 2014
Adenosine deaminase acting on RNA1 (ADAR1) catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in regions of RNA with double-stranded (ds) character. This process is known as A-to-I RNA editing. Alternative promoters drive the expression of the Adar1 gene and alternative splicing gives rise to transcripts that encode 2 ADAR1 protein size isoforms. ADAR1 p150 is an interferon (IFN)-inducible dsRNA adenosine deaminase found in the cytoplasm and nucleus, whereas ADAR1 p110 is constitutively expressed and nuclear in localization. Dependent on the duplex structure of the dsRNA substrate, deamination of adenosine by ADAR can be either highly site-selective or nonspecific. A-to-I editing can alter the stability of RNA structures and the coding of RNA as I is read as G instead of A by ribosomes during mRNA translation and by polymerases during RNA replication. A-to-I editing is of broad physiologic significance. Both the production and the action of IFNs, and hence the subsequent interaction of viruses with their hosts, are among the processes affected by A-to-I editing.
Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site
2002
RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and crosslinking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be crosslinked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2⅐RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.
RNA, 2000
Members of the double-stranded RNA-(dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT 2C R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT 2C R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to singlestranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing.
Activity Regulation of Adenosine Deaminases Acting on RNA (ADARs)
Molecular Neurobiology, 2012
Adenosine deaminases acting on RNA (ADARs) are the enzymes that are responsible for the A to I RNA editing process in mammals, which is an important mechanism that increases molecular diversity. A to I RNA editing consists of an enzymatic conversion of specific adenosine in pre-mRNA, leading to alteration of the properties of both the RNA itself and the translated protein.
Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain
RNA, 2006
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.
Matching Active Site and Substrate Structures for an RNA Editing Reaction
Journal of the American Chemical Society, 2009
The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in a double-stranded structure found in various RNA substrates, including mRNAs. Here we present recent efforts to define structure/activity relationships for the ADAR reaction. We describe the synthesis of new phosphoramidites for the incorporation of 7-substituted-8-aza-7-deazaadenosine derivatives into RNA. These reagents were used to introduce the analogues into mimics of the R/G-editing site found in the pre-mRNA for the human glutamate receptor B subunit (GluR B). Analysis of the kinetics of the ADAR2 reaction with analogue-containing RNAs indicated 8-aza-7-deazaadenosine is an excellent substrate for this enzyme with a deamination rate eight times greater than that for adenosine. However, replacing the C7 hydrogen in this analogue with bromine, iodine, or propargyl alcohol failed to increase the deamination rate further but rather decreased the rate. Modeling of nucleotide binding in the enzyme active site suggested amino acid residues that may be involved in nucleotide recognition. We carried out a functional screen of a library of ADAR2 mutants expressed in S. cerevisiae that varied the identity of these residues to identify active deaminases with altered active sites. One of these mutants (ADAR2 R455A) was able to substantially overcome the inhibitory effect of the bulky C7 substituents (-Br, -I, propargyl alcohol). These results advance our understanding of the importance of functional groups found in the edited nucleotide and the role of specific active site residues of ADAR2.
Journal of Biological Chemistry, 2007
RNA editing that converts adenosine to inosine in doublestranded RNA (dsRNA) is mediated by adenosine deaminases acting on RNA (ADAR). ADAR1 and ADAR2 form respective homodimers, and this association is essential for their enzymatic activities. In this investigation, we set out experiments aiming to determine whether formation of the homodimer complex is mediated by an amino acid interface made through protein-protein interactions of two monomers or via binding of the two subunits to a dsRNA substrate. Point mutations were created in the dsRNA binding domains (dsRBDs) that abolished all RNA binding, as tested for two classes of ADAR ligands, long and short dsRNA. The mutant ADAR dimer complexes were intact, as demonstrated by their ability to co-purify in a sequential affinity-tagged purification and also by their elution at the dimeric fraction position on a size fractionation column. Our results demonstrated ADAR dimerization independent of their binding to dsRNA, establishing the importance of protein-protein interactions for dimer formation. As expected, these mutant ADARs could no longer perform their catalytic function due to the loss in substrate binding. Surprisingly, a chimeric dimer consisting of one RNA binding mutant monomer and a wild type partner still abolished its ability to bind and edit its substrate, indicating that ADAR dimers require two subunits with functional dsRBDs for binding to a dsRNA substrate and then for editing activity to occur. . 2 The abbreviations used are: ADAR, adenosine deaminases acting on RNA; A 3 I, adenosine-to-inosine; dsRNA, double-stranded RNA; dsRBD, dsRNA binding domain; mAb, monoclonal antibody; WT, wild type.
Purification of Native and Recombinant Double-Stranded RNA-Specific Adenosine Deaminases
Methods, 1998
adjacent 3 intron to form a double-stranded (ds) ADAR1 and ADAR2 are members of a family of enzymes RNA structure. Mutations disrupting the base pairthat catalyze the conversion of adenosine to inosine in douing interfere with editing, whereas mutations restorble-stranded RNA. Unlike the other types of RNA editing that ing complementarity also restore editing (5, 6). Inoinvolve multiprotein editing complexes, the site-specific desine is read as guanosine by the translational amination of an adenosine to inosine is catalyzed by single machinery ; therefore, the site-selected deaminaenzymes. ADAR1 and ADAR2 have been purified and the tion of adenosine to inosine changes the amino acid genes cloned from various sources. Each gene encodes multiincorporated at functionally critical sites in the enple splice variants. As it is crucial to have an adequate supply coded proteins (8-10).
Evolutionarily conserved human targets of adenosine to inosine RNA editing
Nucleic Acids Research, 2005
A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice.