Mechanism of the gBP21-mediated RNA/RNA annealing reaction: matchmaking and charge reduction.

The guide RNA-binding protein gBP21 has been characterized as a mitochondrial RNA/RNA annealing factor. The protein co-immunoprecipitates with RNA editing ribonucleoprotein complexes, which suggests that gBP21 contributes its annealing activity to the RNA editing machinery. In support of this view, gBP21 was found to accelerate the hybridization of cognate guide (g)RNA/pre-edited mRNA pairs. Here we analyze the mechanism of the gBP21-mediated RNA annealing reaction. Three possible modes of action are considered: chaperone function, matchmaker function and product stabilization. We conclude that gBP21 works as a matchmaker by binding to gRNAs as one of the two RNA annealing reactants. Three lines of evidence substantiate this model. First, gBP21 and gRNAs form a thermodynamically and kinetically stable complex in a 1 + 1 stoichiometry. Secondly, gRNA-bound gBP21 stabilizes single-stranded RNA, which can be considered the transition state in the annealing reaction. Thirdly, gBP21 has a low affinity for double-stranded RNAs, suggesting the release of the annealed reaction product after the hybridization step. In the process, up to six ionic bonds are formed between gBP21 and a gRNA, which decreases the net negative charge of the RNA. As a consequence, the electrostatic repulsion between the two annealing reactants is reduced favoring the hybridization reaction.