The long term goal of this project is to understand the pathways of RNA turnover in mitochondria. Mitochondrial mRNAs are not capped at the 5' end, nor polyadenylated at the 3' end as cytoplasmic mRNAs are. The nature of the degradation machinery and the protection mechanisms for mitochondrial mRNAs are largely unexplored. It is known that nuclearly encoded factors enter mitochondria to regulate the stability of the mRNAs. A general protection complex binds to the 3' end of all seven yeast mitochondrial mRNAs, while message-specific proteins protect the 5' ends of individual mRNAs.
The Specific Aims of this proposal are: 1) to uncover the general mechanisms of RNA turnover in yeast mitochondria, 2) to determine the role of the Cbp1 protein as a specific regulator of cytochrome b (COB) mRNA processing and turnover, 3) to probe the importance of Cbp1 membrane attachment in mRNA stability and translation, and 4) to identify domains of Cbp1 important for RNA binding and protein interaction. 1) The predominant direction of decay of mRNAs will be determined with the help of strong secondary structures that block exonucleases and trap labile decay intermediates. Wildtype and mutant strains defective in 3' to 5' and 5' to 3' mRNA decay will be assayed. Three new putative turnover components will be characterized. 2) COB pre-mRNA is processed to form the mature 5' end just upstream of the Cbp1 protection site, however we do not know whether the precursor is processed by an exo- or endonucleolytic mechanism. COB mRNA is degraded in the absence of Cbp1, but we do not know if this is because the mRNA is exposed to a degradative exo- or endonuclease. Both of these questions will be answered by using tandem Cbp1 protection sites, one of which is temperature-sensitive, and if monitoring the stability of the RNA and the position of the 5' ends. 3) Cbp1 like all mitochondrial message-specific factors, is attached to the inner mitochondrial membrane, but it is not known how it is attached or whether attachment is required for mRNA stability and translation. Membrane attachment will be probed by a variety of genetic methods. 4) Cbp1 protects COB mRNA. Genetic methods will be used to define domains of Cbp1 involved in RNA-binding and contacts with other proteins, as a prelude to the development of in vitro assays. Several different human disease syndromes and effects of aging are due to mitochondrial dysfunction. These syndromes are due to mutations in mitochondrial DNA and nuclear genes encoding mitochondrial proteins. It is likely that general themes true for yeast mitochondria will in some cases prove true for mammalian mitochondria as well. The proposed work will elucidate an unexplored dimension of metabolism of eukaryotic cells in general.
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