The proposed work centers on determining the role RNA processing plays for the regulation of mitochondrial gene expression in Saccharomyces cerevisiae. Previous studies showed that processing and splicing of the pre-21S rRNA require no mitochondrial encoded proteins. Thus, any enzymes which catalyze these reactions must be encoded by nuclear genes. Unique to yeast, however, is the ability to isolate and genetically manipulate mutants in both genomes, i.e., mutants in the mitochondrial encoded RNA substrates and in the nuclear encoded enzymes. In part, the aim of the proposed studies is to select such mutants, particularly those mapping to the nucleus which block splicing of the pre-21S rRNA. A novel screen has been employed to isolate several nuclear mutants that potentially are deficient in the splicing or processing of the mosaic pre-21S rRNAs found in the mitochondrion. The isolated mutants are respiratory-deficient when harboring the intron plus allele of that gene, but are phenotypically wild-type when containing the intron-less allele. Among a collection of such nuclear mutants ought to be found mutations in the genes encoding enzymes or structural proteins required for RNA processing. Mutants in both the nuclear and mitochondrial genomes that block expression of the mitochondrial 21S rRNA gene will provide tools for the characterization of mitochondrial RNA processing in molecular terms. Determining the elements of RNA sequence and structure and the enzymatic requirements for site-specific RNA processing will be investigated by in vitro transcription assays using mitochondria isolated from wild-types and mutants. Mapping of RNAs accumulated in these strains will be accomplished by S1 protection experiments, sequencing of cDNAs, and northern blot analyses. In addition, an in vitro assay for splicing has been developed using ribonucleoprotein particles prepared from either petite or wild-type mitochondria. This assay will be applied to the analysis of splicing in the mutants in order to determine at what step in the processing pathway the mutants are blocked. In the long term, these studies will permit the isolation, by complementation cloning in yeast, of the nuclear genes encoding enzymes for mitochondrial RNA processing. In part, the regulation of mitochondrial biogenesis must include control over the expression of such nuclear genes.
