| Congresso Brasileiro de Microbiologia 2023 | Resumo: 964-1 | ||||
Resumo:Background: Mitochondrial RNase P of the yeast Saccharomyces is responsible for endonucleolytic cleavage of the 5’ leader sequences of mitochondrial tRNA precursor transcripts. This RNase P comprises a catalytic RNA, RPM1, and a protein subunit, Rpm2. Rpm2 is encoded by a nuclear gene and imported from the cytoplasm after synthesis, while RPM1 RNA is transcribed from the mitochondrial genome along with two flanking tRNAs. Processing RPM1 to the mature form requires the activity of the RNase P itself, an interesting positive feedback loop. We previously discovered that mitochondrial fatty acid biosynthesis is required for the efficient processing of the precursor RPM1 RNA but did not know why. Our current hypothesis is that fatty acid modification is necessary for full RNase P function via assembly with other proteins in a complex and/or attachment to the mitochondrial membrane.
Methods and Results: We have now determined that the Rpm2 protein is covalently modified by a fatty acid using a gel mobility shift assay to compare wildtype and mitochondrial fatty acid biosynthesis mutant strains. By deletion analysis we have localized the modification to the terminal 259 residues of the 1202 residue protein. The fine mapping of the fatty acid attachment site on Rpm2 is ongoing, primarily through site-directed mutagenesis of Lysine residues (preferred for covalent fatty acid modification) in the C-terminal region, followed by growth assays of mitochondrial function on medium requiring utilization of the nonfermentable carbon source glycerol and gel shift analysis of fatty acid attachment to the C-terminal Rpm2 fragment by Western blotting of mitochondrial extracts. Isogenic wildtype and fatty acid synthesis mutant strains are used as positive and negative controls. The covalently attached fatty acid will be identified, and the attachment site verified, by mass spectrometry. In addition, previous genetic and pulldown studies showed that Rpm2 is part of a multimeric complex. This complex has been found also by others recently as part of a comprehensive blue native gel analysis followed by mass spectrometry of all yeast mitochondrial protein complexes. Our colleague, Kaija Autio, U Oulu, Finland, has preliminary mass spectrometry data supporting the multimeric composition of the complex. We are continuing further genetic, biochemical, structural, and functional analyses of the complex. Palavras-chave: mitochondria, fatty acid biosynthesis, 5' tRNA processing, protein modification, Saccharomyces cerevisiae Agência de fomento:FAPESP, CAPES | |||||