Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding.
Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs.
Macbeth
Mark R
MR
Howard Hughes Medical Institute and Department of Biochemistry, University of Utah, Salt Lake City, UT 84132, USA.
Lingam
Arunth T
AT
Bass
Brenda L
BL
D000243:Adenosine Deaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058892:Adenosine Deaminase Inhibitors; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D058892:Adenosine Deaminase Inhibitors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase