In Vitro Characterization Of The Splicing Of The Terminal Intron Of The Mitochondrial Apocytochrome B Gene Of Saccharomyces Cerevisiae PDF Download

Hypothesized to be ancestors of eukaryotic spliceosomal introns, extant group II introns are found in bacteria, archaea, and in the mitochondria and chloroplast genomes of some eukaryotes. While some of these catalytic intron RNAs encode an intron-encoded maturase to assist in their splicing, not all do, eliciting the question of how these introns are spliced out. Two such introns are the mitochondrial introns aI5 [gamma] and bI1 of Saccharomyces cerevisiae. Previous in vitro studies on the self-splicing activity of these introns revealed requirements for non-physiologically high salt concentration and temperature for catalytic activity, suggesting a dependence upon proteins in vivo. With most mitochondrial proteins known to be encoded by the nucleus, it seems likely that some exist to assist, either directly or indirectly, in the correct splicing of these introns. Previous searches for such proteins relied on a combination of two genetic screens: looking for a mutant's inability to grow on the non-fermentable carbon source glycerol-(Gly− phenotype)- in the presence of a given intron, and a Gly+ phenotype in the intron's absence. In contrast, this study employs Northern hybridization to identify splicing defects in a series of mutants constructed by targeted gene deletion, allowing for identification of proteins required for splicing that would not have been otherwise detectable. In order to identify associated proteins, this method is complemented by Tandem Affinity Purification of Mss116, the DEAD-box RNA chaperone required for splicing of all the yeast mitochondrial introns. In addition to Mss116, special attention was given to the insertase Oxa1, found to be required for aI5 [gamma] and bI1splicing specifically; and to Mne1, found to be required for splicing group I intron aI5 [beta]. The results point to distinctly different auxiliary protein requirements for group I introns, group II introns that encode their own maturases, and group II introns that do not encode maturases. My findings are consistent with the possibility that the splicing of aI5 [gamma] and bI1, group II introns that do not encode maturases, is coordinated by multiple proteins comprising a splicing complex situated at the surface of the mitochondrial inner membrane in close association with the mitoribosomes.