The long-term goal of the proposed research is to better understand host cell-organelle interactions. Specifically this grant addresses the role of nuclear-coded proteins in the expression of the cytochrome b, COB, mitochondrial mRNA in Saccharomyces cerevisiae. cbp1p interacts with the distal portion of the untranslated leader of COB mRNA to promote stability of the message, while cbs1p and Cbs2p interact with the proximal portion of the leader to promote translation.
The Specific Aims of this proposal are: 1) To identify proteins that interact with Cbp1p and COB mRNA to promote stability of the mRNA via reversion analyses, a dihybrid screen and biochemical analyses. 2) To determine the sequence element and/or secondary structures that are important in the region of COB mRNA that interacts with cbplp via site-directed mutagenesis and a physical analysis of the in vivo structure of the RNA. 3) To determine the directionality of decay of COB mRNA in vivo to begin to elucidate the mechanism of turnover. An RNA structure that is resistant to exonucleolytic decay will be inserted at intervals in the mRNA to investigate whether the message is primarily degraded by 5' to 3' or 3' to 5' exonucleases or 5'-dependent endonucleases. 4) To determine the sequence element and/or secondary structures that are important in the regions of COB mRNA that are required for translation via linker-scanning mutagenesis and physical analysis of the in vivo structure of the RNA. These studies will expand the understanding of RNA-protein interactions specifically and in general will contribute to furthering the knowledge of organelle function in eukaryotic cells. Defects in mitochondrial function have been implicated in muscle wasting diseases, aging and lethal genetically inherited disorders. Thus, it is important that the details of nuclear control of mitochondrial function be elucidated.
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