A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria.

The Pet54p protein is an archetypical example of a dual functioning ('moonlighting') protein: it is required for translational activation of the COX3 mRNA and splicing of the aI5beta group I intron in the COX1 pre-mRNA in Saccharomyces cerevisiae mitochondria (mt). Genetic and biochemical analyses in yeast are consistent with Pet54p forming a complex with other translational activators that, in an unknown way, associates with the 5' untranslated leader (UTL) of COX3 mRNA. Likewise, genetic analysis suggests that Pet54p along with another distinct set of proteins facilitate splicing of the aI5beta intron, but the function of Pet54 is, also, obscure. In particular, it remains unknown whether Pet54p is a primary RNA-binding protein that specifically recognizes the 5' UTL and intron RNAs or whether its functional specificity is governed in other ways. Using recombinant protein, we show that Pet54p binds with high specificity and affinity to the aI5beta intron and facilitates exon ligation in vitro. In addition, Pet54p binds with similar affinity to the COX3 5' UTL RNA. Competition experiments show that the COX3 5'UTL and aI5beta intron RNAs bind to the same or overlapping surface on Pet54p. Delineation of the Pet54p-binding sites by RNA deletions and RNase footprinting show that Pet54p binds across a similar length sequence in both RNAs. Alignment of the sequences shows significant (56%) similarity and overlap between the binding sites. Given that its role in splicing is likely an acquired function, these data support a model in which Pet54p's splicing function may have resulted from a fortuitous association with the aI5beta intron. This association may have lead to the selection of Pet54p variants that increased the efficiency of aI5beta splicing and provided a possible means to coregulate COX1 and COX3 expression.

An allosteric-feedback mechanism for protein-assisted group I intron splicing.

The I-AniI maturase facilitates self-splicing of a mitochondrial group I intron in Aspergillus nidulans. Binding occurs in at least two steps: first, a specific but labile encounter complex rapidly forms and then this intermediate is slowly resolved into a native, catalytically active RNA/protein complex. Here we probe the structure of the RNA throughout the assembly pathway. Although inherently unstable, the intron core, when bound by I-AniI, undergoes rapid folding to a near-native state in the encounter complex. The next transition includes the slow destabilization and docking into the core of the peripheral stacked helix that contains the 5' splice site. Mutational analyses confirm that both transitions are important for native complex formation. We propose that protein-driven destabilization and docking of the peripheral stacked helix lead to subtle changes in the I-AniI binding site that facilitate native complex formation. These results support an allosteric-feedback mechanism of RNA-protein recognition in which proteins engaged in an intermediate complex can influence RNA structure far from their binding sites. The linkage of these changes to stable binding ensures that the protein and RNA do not get sequestered in nonfunctional complexes.