Adenosine to Inosine Editing by ADAR2 Requires Formation of a Ternary Complex on the GluR-B R/G Site

Abstract

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 crosslinking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be crosslinked 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 ADAR2⅐RNA 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.Eukaryotic precursor-mRNAs (pre-mRNAs) are subject to a variety of post-transcriptional modifications that regulate gene expression. These modifications, collectively referred to as RNA processing reactions, include 5Ј-and 3Ј-end formation, RNA splicing, polyadenylation, and the recently characterized phenomenon of RNA editing (reviewed in Refs.

Biochemical analysis and scanning force microscopy reveal productive and nonproductive ADAR2 binding to RNA substrates

“…This is in agreement with the observation in gel mobility assays that are indicative for a stepwise formation of a monomer and then a dimer at an editing site (see Fig. 2C; Jaikaran et al 2002).…”

“…As previously seen, the short substrate containing only the R/G stem-loop exhibited two defined complex bands ( Fig. 2C), which have been suggested to represent binding of protein monomers and dimers (Jaikaran et al 2002).…”

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

“…Most of the evidence indicates that the answer is yes. First, mobility shift assays have demonstrated consecutive binding of two ADAR2 monomers to an RNA (Ohman et al 2000;Jaikaran et al 2002). Second, high concentrations of RNA inhibit editing suggesting that excess of RNA leaves no enzymes free in solution for dimerization (Hough and Bass 1994).…”

“…The precise mechanism of how the RNA binding facilitates catalysis is still uncertain, but in vitro studies suggest that formation of a ternary complex with two ADAR2 proteins per substrate is required for activity (Jaikaran et al 2002), and purifications of ADAR1 and 2 indicate that they both form homodimers (Cho et al 2003). For the Drosophila ADAR, the N terminus and the first DRBM were shown to be necessary for dimerization and catalytic activity (Gallo et al 2003), while the human ADAR2 does not depend on the N-terminal region for RNA editing (Macbeth et al 2004), suggesting that the ADAR2 dimerization domain differs from that of the Drosophila ADAR.…”

Molecular dynamics simulations and free energy calculations of base flipping in dsRNA

“…Since inosine is recognized as guanosine at translation, editing within a coding sequence will often lead to a change in codon usage. Evidence has been found that ADARs form homodimers on dsRNA, a necessary event for editing activity (Jaikaran et al 2002;Cho et al 2003;Gallo et al 2003). ADAR enzymes deaminate adenosines nonselectively within completely base-paired dsRNA that is longer than 50 bp (Nishikura et al 1991;Polson and Bass 1994).…”