RNA Binding-independent Dimerization of Adenosine Deaminases Acting on RNA and Dominant Negative Effects of Nonfunctional Subunits on Dimer Functions

Abstract

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. RNA editing mediated by adenosine deaminases acting on RNA (ADAR)2 involves adenosine-to-inosine (A 3 I) changes in double-stranded RNA (dsRNA) (1-4). A 3 I RNA editing can alter the protein coding sequence of several genes to create various isoforms, such as in the glutamate receptor ion channel subunits (5, 6), serotonin 2C subtype receptors (7), and Kv1.1 potassium channel (8). The association of malfunctioning A 3 I editing mechanisms and certain human disease, such as neurodegeneration in amyotrophic lateral sclerosis and depression in suicide victims has been implicated (9, 10). Bioinformatic approaches have revealed numerous A 3 I editing sites within non-coding sequences in introns and untranslated regions harboring repetitive elements such as . Furthermore, recent evidence has revealed the intersection of ADAR with the RNA interference pathway, indicating a much broader role for A 3 I RNA editing (16 -21). A 3 I RNA editing is mediated by ADAR (1-4). In vertebrates, three separate ADAR family members have been identified, and they are conserved in their C-terminal deaminase region as well as in their N-terminal double-stranded RNA binding domains (dsRBDs) (22)(23)(24)(25)(26)(27)(28)(29). ADARs are also present in invertebrates, such as a single Drosophila member (dADAR) that is similar to the mammalian ADAR2 (30), as well as two less conserved Caenorhabditis elegans members (c.e.ADR1 and c.e.ADR2) (22, 31).The common structural features shared by mammalian ADARs include dsRBDs repeated two or three times that are located in the N-terminal region...

Adenosine Deaminase That Acts on RNA 3 (ADAR3) Binding to Glutamate Receptor Subunit B Pre-mRNA Inhibits RNA Editing in Glioblastoma

“…Previous studies of ADAR3 demonstrated that mutation of these lysine residues to glutamate (E) and alanine (A) disrupts dsRNA binding in vitro (60). Western blotting of U87 cells transduced with retroviruses expressing the ADAR3 mutants driven by the CMV promoter resulted in similar expression levels as the U87 cells stably expressing wildtype ADAR3 driven by the CMV promoter (Fig.…”

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

“…Consistent with its nuclear localization, ADAR1 interacted with endogenous DGCR8 (Figure 5B), but its interaction with DICER was barely detectable under our experimental conditions. In addition, both the catalytically inactive ADAR1 mutant (E912A) [22,50] and the RNA-bindingnull ADAR1 mutant (EAA) [51] acted in a similar manner in the association with DGCR8 ( Figure 5B and data not shown), indicating that these ADAR1 mutations do not affect its association with DGCR8.…”

Adenosine Deaminases Acting on RNA, RNA Editing, and Interferon Action

“…O-phenanthroline, a chelator of zinc ions, inhibits the enzymatic activity of purified natural ADAR1 (Kim and others 1994a). The catalytically active form of ADAR1 is a dimer (Cho and others 2003;Gallo and others 2003;Valente and Nishikura 2007). Fluorescence resonance energy transfer (FRET) and immunoprecipitation analyses have further demonstrated that both ADAR1 and ADAR2 form homodimers in human cells, that ADAR1 and ADAR2 may also form heterodimers, and that the dimerization likely is RNA-independent (Chilibeck and others 2006;Cenci and others 2008).…”