title: Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2.
abstract: Adenosine deaminases acting on RNA (ADARs) hydrolytically deaminate adenosines (A) in a wide variety of duplex RNAs and misregulation of editing is correlated with human disease. However, our understanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic domain. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interactions. Here we use a recently discovered RNA substrate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucleoside analog 8-azanebularine (8-azaN) into this RNA (and derived constructs) to mechanistically trap the protein-RNA complex without catalytic turnover for EMSA and ribonuclease footprinting analyses. EMSA showed that hADAR2-D requires duplex RNA and is sensitive to 2'-deoxy substitution at nucleotides opposite the editing site, the local sequence and 8-azaN nucleotide positioning on the duplex. Ribonuclease V1 footprinting shows that hADAR2-D protects ∼ 23 nt on the edited strand around the editing site in an asymmetric fashion (∼ 18 nt on the 5' side and ∼ 5 nt on the 3' side). These studies provide a deeper understanding of the ADAR catalytic domain-RNA interaction and new tools for biophysical analysis of ADAR-RNA complexes.
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Phelps
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Kiet
authors.initials: K
authors.lastname: Tran
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Tristan
authors.initials: T
authors.lastname: Eifler
authors.affiliation: Department of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Anna I
authors.initials: AI
authors.lastname: Erickson
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA Department of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Fisher
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA pabeal@ucdavis.edu.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Beal
mesh_terms: D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011684:Purine Nucleosides / Q000737:chemistry; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012263:Ribonucleosides / Q000737:chemistry
chemical_list: C520167:8-azanebularine; D011684:Purine Nucleosides; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D012263:Ribonucleosides; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase