Chimeric double-stranded RNA-specific adenosine deaminase ADAR1 proteins reveal functional selectivity of double-stranded RNA-binding domains from ADAR1 and protein kinase PKR.
The RNA-specific adenosine deaminase (ADAR1) and the RNA-dependent protein kinase (PKR) are both interferon-inducible double-stranded (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 R/G and (+)60 sites of the glutamate receptor B subunit pre-RNA and completely abolished editing of the serotonin 2C receptor (5-HT(2C)R) 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(2C)R 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.
Liu
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Department of Molecular, Cellular and Developmental Biology, and Interdepartmental Graduate Program of Biochemistry and Molecular Biology, University of California, Santa Barbara, CA 93106, USA.
Lei
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Samuel
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D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D003641:Deamination; D006801:Humans; D017434:Protein Structure, Tertiary; D017393:RNA Editing; D012322:RNA Precursors / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins; D044405:Receptor, Serotonin, 5-HT2C; D017470:Receptors, Glutamate / Q000235:genetics*; D011985:Receptors, Serotonin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D012322:RNA Precursors; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D044405:Receptor, Serotonin, 5-HT2C; D017470:Receptors, Glutamate; D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins; D019892:eIF-2 Kinase; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D000241:Adenosine