Mitochondrial tRNA processing complex

• Grant number: 23/02534-4
• Support Opportunities: Research Grants - Visiting Researcher Grant - International
• Start date: September 05, 2023
• End date: November 20, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Mario Henrique de Barros
• Grantee: Mario Henrique de Barros
• Visiting researcher: Carol Louise Dieckmann
• Visiting researcher institution: University of Arizona, United States
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Mitochondria are essential for life; they are critical subcellular compartments that generate the power driving most cellular reactions. We use the common model organism, baker's yeast, to study the genetics and biochemistry of mitochondrial function. Yeast, like all eukaryotes, has an 80,000 base pair mitochondrial genome in addition to 12 million base pairs in nuclear chromosomes. Transcription of the mitochondrial genome produces multigenic precursor RNAs that must be processed before they function as mRNAs, rRNAs and tRNAs. We propose to focus the research in this proposal on the yeast mitochondrial RNase P enzyme, which cleaves precursor tRNAs to produce mature 5' ends. Dr. Dieckmann's lab has shown that the Rpm2 protein component of this RNase P enzyme is attached to a fatty acid, a covalent modification required for activity. Dr. Barros' lab has shown that Rpm2 resides in a complex of several additional proteins also required for 5' tRNA processing and that the complex is associated with the inner mitochondrial membrane. Our current hypothesis is that the fatty acid modification of Rpm2 allows anchoring of the enzyme in the inner mitochondrial membrane and/or assembly with other proteins necessary for optimal RNase P function in some way that we still do not understand. The goal of the research is to determine whether fatty acid modification of Rpm2 is necessary for RNase P membrane association and/or formation of a complex with other proteins, and the effect on RNase P activity in yeast mitochondria. Objective 1: Separate mutations in a temperature sensitive (ts) Rpm2 mutant. Dr. Barros made collections of ts mutations in the genes coding for five proteins required for tRNA processing. In one ts strain, there are three mutations in the gene for Rpm2. We will make three strains with each individual mutation and a double mutant strain with two nearby mutations. All mutations will be made by site-directed mutagenesis and the gene will be re-introduced into a strain with no wildtype Rpm2. Objective 2: Analyze the effect of the mutations on respiratory growth and fatty acid modification of Rpm2. Growth of yeast colonies will be assessed on plates requiring respiration. Fatty acid modification of Rpm2 will be assessed by gel shift assay of immunoblotted CH-tagged Rpm2. Objective 3: Determine whether the ts mutations disturb the formation of the large complex of all seven proteins, affect the interaction of Rpm2 and Mta1 specifically, and/or affect association with the membrane. The effects of mutations on the large complex can be assessed by blue-native gel electrophoresis followed by immunoblotting with anti-tag antisera (different tags on Rpm2 and Mta1). Interaction of Rpm2 and Mta1 will be assessed by pull downs with anti-tag beads from extracts of a strain tagged on Rpm2, followed by immunoblotting with the respective tag antisera. Reciprocal assessments will be done starting with Mta1 pulldowns. Membrane association will be assessed by a combination of differential centrifugation, and salt and/or detergent extraction. Objective 4: Determine whether knocking out mitochondrial fatty acid synthesis (KO mutation of the gene encoding Etr1, which encodes one enzyme in the biosynthetic pathway) affects large complex formation, Rpm2-Mta1 interaction, and/or membrane association. Significance: Our work will further the knowledge of the multi-subunit 5' tRNA processing enzyme complex and the crucial requirement for covalent fatty acid modification of Rpm2. This may prove to be a model for the regulation of other mitochondrial protein assemblies. (AU)