Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity

Abstract

ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein

“…This result indicates that much if not most of this neighboring sequence preference derives from the ADARcd, despite an indication that the 3 ′ guanosine preference might derive from the ADAR dsRBDs (Stefl et al 2010). Although binding could be sequential, i.e., first dsRBD recognition followed by a conformational change and ADARcd recognition, the ADARcd appears to contain most of the specificity (Matthews et al 2016).…”

“…As previously discussed (Kuttan and Bass 2012;Matthews et al 2016), the E488Q mutation either enhances base flipping, perhaps by enhanced amino acid insertion by the HyperTRIBE ADARcd into the RNA A helix, or it has a much higher rate of successful editing/flipping events. Notably, the ADARcd loop that occupies the displaced A during flipping (Matthews et al 2016) contains amino acid 488.…”

“…It preferentially edits adenosines bordered by 5 ′ uridines and 3 ′ guanosines, i.e., a UAG sequence (Lehmann and Bass 2000;Eggington et al 2011), which is also observed in TRIBE (McMahon et al 2016). The ADARcd also prefers to edit adenosines surrounded by a double-stranded region (Bass and Weintraub 1988;Kim et al 1994;O'Connell et al 1995;Eggington et al 2011;Matthews et al 2016). Consequently, a substantial fraction of RBP-target mRNAs may go unidentified.…”

“…Notably, the ADARcd loop that occupies the displaced A during flipping (Matthews et al 2016) contains amino acid 488. Better binding of the HyperTRIBE ADARcd to its substrate region might enhance the transition state lifetime and therefore editing efficiency (Kuttan and Bass 2012;Matthews et al 2016). This is further suggested by the impact of distance from the CLIP sites on editing efficiency as well as on the structural requirement surrounding the edited adenosine (Fig.…”

Adenosine Deaminase That Acts on RNA 3 (ADAR3) Binding to Glutamate Receptor Subunit B Pre-mRNA Inhibits RNA Editing in Glioblastoma

“…Our data indicate that the deaminase domain does not function in ADAR3-mediated regulation of GRIA2 editing, raising questions about the functional significance of the domain. Interestingly, a recent structural study identified a key arginine residue in the RNA binding loop of the deaminase domain that is conserved in ADAR2 and ADAR1 but is a glutamine in ADAR3 (74). As mutation of this arginine to glutamine in human ADAR2 reduced editing activity 10-fold, these data provide an explanation for the lack of deamination activity observed for ADAR3.…”