Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site.
RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and cross-linking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be cross-linked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2.RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.
Jaikaran
Dominic C J
DC
Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.
Collins
Cynthia H
CH
MacMillan
Andrew M
AM
D000241:Adenosine / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D006801:Humans; D007288:Inosine / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017393:RNA Editing / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018091:Receptors, AMPA; D011994:Recombinant Proteins; C115953:glutamate receptor type B; D007288:Inosine; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D000241:Adenosine