Chimeric double-stranded RNA-specific adenosine deaminase ADAR1 proteins reveal functional selectivity of double-stranded RNA-binding domains from ADAR1 and protein kinase PKR

Abstract

The RNA-specific adenosine deaminase (ADAR1) and the RNA-dependent protein kinase (PKR) are both interferon-inducible doublestranded (ds) RNA-binding proteins. ADAR1, an RNA editing enzyme that converts adenosine to inosine, possesses three copies of a dsRNA-binding motif (dsRBM). PKR, a regulator of translation, has two copies of the highly conserved dsRBM motif. To assess the functional selectivity of the dsRBM motifs in ADAR1, we constructed and characterized chimeric proteins in which the dsRBMs of ADAR1 were substituted with those of PKR. Recombinant PKR-ADAR1 chimeras retained significant RNA adenosine deaminase activity measured with a synthetic dsRNA substrate when the spacer region between the RNA-binding and catalytic domains of the deaminase was exactly preserved. However, with natural substrates, substitution of the first two dsRBMs of ADAR1 with those from PKR dramatically reduced site-selective editing activity at the R͞G and (؉)60 sites of the glutamate receptor B subunit pre-RNA and completely abolished editing of the serotonin 2C receptor (5-HT 2CR) pre-RNA at the A site. Chimeric deaminases possessing only the two dsRBMs from PKR were incapable of editing either glutamate receptor B subunit or 5-HT 2CR natural sites but edited synthetic dsRNA. Finally, RNA antagonists of PKR significantly inhibited the activity of chimeric PKR-ADAR1 proteins relative to wild-type ADAR1, further demonstrating the functional selectivity of the dsRBM motifs.T he RNA-specific adenosine deaminases (ADAR) (1) constitute a multigene family of editing enzymes. They catalyze the C-6 deamination of adenosine to produce inosine in double-stranded (ds) structures present within cellular pre-RNAs and viral RNAs as well as synthetic dsRNA substrates (2-6). So far, two functional RNA adenosine deaminases, denoted ADAR1 and ADAR2, have been described (1, 6). We isolated molecular cDNA clones of ADAR1 as an interferon-inducible enzyme (7,8). The ADAR1 protein possesses both dsRNA-binding and Z-DNA-binding properties (8)(9)(10)(11)(12)(13)(14). The dsRNA-binding domain of the 1,226-aa ORF of the ADAR1 cDNA consists of three copies of the highly conserved dsRNA-binding motif (dsRBM). The prototype dsRBM including the R core amino acid residues was first identified in the interferoninducible RNA-dependent protein kinase PKR, an enzyme that plays a pivotal role in the antiviral actions of IFN and the control of translation in virus-infected cells (15,16). PKR possesses two copies of the dsRBM that cannot substitute for each other (17)(18)(19)(20). RNAs bound by PKR via the dsRBMs mediate the autophosphorylation and activation of PKR (19,21), thereby leading to the subsequent phosphorylation of the ␣ subunit of protein synthesis initiation factor eIF-2 (22, 23).Three naturally occurring splice variant isoforms of human ADAR1 are known that are differentially expressed in different tissues (24). In comparison to the full-length 1,226-aa ADAR1 protein (8-10), denoted ADAR1-a, the variant designated ADAR1-b possesses a deletion of 26 a...

Interactions between the double-stranded RNA-binding proteins TRBP and PACT define the Medipal domain that mediates protein-protein interactions

“…50 With the exception of DIP1 dsRBD2, all dsRBDs that bind dsRNA are of type A. This has been demonstrated for Staufen, 44,51 TRBP, 24 XlRBPA, 52 PACT, 40 PKR, 49 ADAR, 53 RHA, 54 E3L, 55 RNase III, 56 and others with various intensities. In contrast, all dsRBDs type B that have been assayed show no RNA binding, 23 ,40,44,49,51,52 although they can cooperate with the other type for stronger RNA binding.…”

Antiviral Actions of Interferons

“…Mutational analyses show that the dsRBM III motif of ADAR1 is essential for deaminase activity whereas RII is dispensable (217,237,240). Chimeric proteins in which the dsRBM of ADAR1 are replaced with those of PKR retain deaminase activity with synthetic dsRNA substrates but show dramatically reduced editing activity with natural RNA substrates (241). Furthermore, VAI and aptamer RNA antagonists of PKR significantly inhibit the deaminase activity of chimeric PKR-ADAR1 proteins but less so of wild-type ADAR1 (241).…”

“…Modulation of eIF-2␣ phosphorylation is the primary function of viral VAI RNA, at least in cell culture, because replacement of serine 51 by alanine not only eliminates the phosphorylation site on the eIF-2␣ substrate but also functionally complements the deletion of VA from the virus genome. Howerver, VAI RNA does antagonize ADAR1 (225,241) in addition to PKR (197,264). EBV-encoded EBER RNAs, which functionally can substitute for adenovirus VAI RNA in vivo, also inhibit PKR activation (68).…”

“…Chimeric proteins in which the dsRBM of ADAR1 are replaced with those of PKR retain deaminase activity with synthetic dsRNA substrates but show dramatically reduced editing activity with natural RNA substrates (241). Furthermore, VAI and aptamer RNA antagonists of PKR significantly inhibit the deaminase activity of chimeric PKR-ADAR1 proteins but less so of wild-type ADAR1 (241). These results indicate that the dsRNA-binding motifs are functionally distinct from each other, as measured by binding substrates in a manner recognized by the enzyme catalytic center for deamination.…”

RNA aptamers binding the double-stranded RNA-binding domain

“…If, for instance, the deaminase domains of ADAR1 and ADAR2 are exchanged, the resulting chimeric enzymes predominantly retain the substrate specificity determined by the deaminase domain (Wong et al 2001). On the other hand, the exchange of dsRBDs of ADAR1 with the ones from PKR leads to a dramatic reduction in substrate recognition of the chimeric protein (Liu et al 2000). Similarly, in Xenopus laevis oocytes it could be shown that Xenopus ADAR1 localizes to transcriptionally active lampbrush chromosomes and binds to the majority of nascent transcripts.…”