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 asse ss the functional selectivity of the dsRBM motifs in ADAR1, we constructed and characterized chimeric proteins in which the dsRBMs of ADAR1 were subst ituted with those of PKR. Recombinant PKR-ADAR1 chimeras retained significa nt RNA adenosine deaminase activity measured with a synthetic dsRNA substra te when the spacer region between the RNA-binding and catalytic domains of the deaminase was exactly preserved. However, with natural substrates, subs titution 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 g lutamate receptor B subunit pre-RNA and completely abolished editing of the serotonin 2C receptor (5-HT2CR) pre-RNA at the A site. Chimeric deaminases possessing only the two dsRBMs from PKR were incapable of editing either g lutamate receptor B subunit or 5-HT2CR 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 demons trating the functional selectivity of the dsRBM motifs.