Nucleic Acids Research Advance Access published online on April 3, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn045
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A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria
Center for RNA Molecular Biology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106-4960, USA
*To whom correspondence should be addressed. Tel: +216 368 4757; Fax: +216 368 2010; Email: mgc3{at}po.cwru.edu
Received November 30, 2007. Revised January 23, 2008. Accepted January 24, 2008.
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 aI5β 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 aI5β 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 aI5β 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 aI5β 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 aI5β intron. This association may have lead to the selection of Pet54p variants that increased the efficiency of aI5β splicing and provided a possible means to coregulate COX1 and COX3 expression.