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Abstract
ADAR2 is an RNA editing enzyme that deaminates adenosines in the context of duplex RNA. The mechanism for its substrate selectivity has not been fully elucidated. The focus of this investigation is to examine the contributions from ADAR2's RNA binding domain that lead to specific RNA editing.
Model RNA substrates, derived from two ADAR2 editing sites in the GluR-B subunit pre-mRNA, were developed so that editing could be monitored in relation to systematic changes in the sequence and structure of the RNA. These short RNA duplexes direct editing to only the adenosines that are deaminated in vivo, indicating that they appropriately mimic their respective natural editing sites. Duplex structure 5′ to the editing site accelerates the rate of deamination. Base bulges naturally found in the structure of an ADAR2 substrate did not appear to direct deamination to the editing site.
Using directed hydroxyl radical cleavage, selective binding by ADAR2's double-stranded RNA binding motifs (dsRBMs) was observed on RNAs that mimic ADAR2 editing sites. With these cleavage data, molecular models were developed that predict two important binding surfaces on the RNA for ADAR2. Blocking these surfaces with site-specific benzyl modification at guanosine 2-amino groups impedes RNA-editing, demonstrating a correlation between deamination efficiency by ADAR2 and selective binding by its dsRBMs. A truncated mutant of ADAR2 that lacks dsRBMI is sensitive to only one of these modified binding surfaces. These data identify the binding site for each ADAR2 dsRBM in the catalytically relevant complex leading to deamination by ADAR2.
It was also observed that the binding selectivity displayed by ADAR2's dsRBMs is different from that of PKR's dsRBMI. This supports the hypothesis that dsRBMs from different proteins possess intrinsic binding selectivity, which influences the parent protein's substrate specificity. This study was the first direct comparison of the binding selectivity for two homologous dsRBMs.
Finally, a 6-trifluoromethylpurine ribonucleoside was designed to be an inhibitor for the ADAR2 deamination reaction. This purine analogue did not display inhibitory effects as a free ribonucleoside or in the context of RNA. However, due to the presence of fluorine, this compound could serve as a 19F NMR probe for future studies.
