Characterization of the Saccharomyces cerevisiae gene MGP1, a novel member of the ras gene superfamily that functions in the mitochondrial biogenesis

This dissertation characterizes Mgp1p, a mitochondrial protein that functions in mitochondrial biogenesis. Mgp1p is the first protein related to the p21[superscript] H-ras oncoprotein shown to function in mitochondria;The Saccharomyces cerevisiae gene MGP1 was isolated based on its ability to restore respiratory competence to a strain containing the nuclear petite mutation msg1-1. MGP1 codes for a protein of approximately 58 kDa, with an amino terminal sequence typical of a mitochondrial targeting peptide. A 190 residue region of Mgp1p shares approximately 30% amino acid sequence identity with any member of p21[superscript] ras-related protein superfamily, with particularly strong homology in the regions known to interact with guanine nucleotides. Multiple copies of MGP1 suppress the respiratory enzyme assembly defects caused by msg1 mutations. To demonstrate that Mgp1p is functionally similar to p21[superscript] ras-related proteins, a mutation was introduced into MGP1 that deleted the phosphate group binding region conserved in this family of guanine nucleotide binding proteins. This mutation inactivated the suppressor function of MGP1;Mgp1p co-fractionated with mitochondria in subcellular fractionation experiments. In vitro mitochondrial import experiments demonstrated Mgp1p is translocated into mitochondria by a mechanism dependent on inner membrane potential, and Mgp1p was processed during translocation. After translocation Mgp1p was bound to mitochondrial membranes. Mutation of cysteine residue 523 to a serine reduced the association of Mgp1p with mitochondrial membranes, suggesting C-terminal post-translational modifications occur for Mgp1p similar to those known for other p21[superscript] ras-related proteins. This mutation inactivated the suppressor function of MGP1;Mgp1p was produced in E. coli to facilitate biochemical analysis. Crosslinking experiments demonstrated Mgp1p expressed in E. coli is capable of binding GTP. Taken together, the data presented in this dissertation suggest Mgp1p is a p21[superscript] ras-related, GTP-binding protein attached to the mitochondrial membrane by a mechanism involving modification at its carboxyl terminus. Based on the known functions of such proteins, Mgp1p may serve as part of a signal transduction mechanism involved in communication between mitochondria and the cytosol

NEGATIVE REGULATORY FACTORS IN TOMBUSVIRUS REPLICATION: KNOWN PROTEINS, NOVEL ROLES

Although host cells are a rather rich source for co-opted host factors, lipids and metabolites, positive stranded RNA viruses vastly rewire cellular pathways and remodel cellular membranes to support viral replication. To accomplish such major changes, these viruses depend on the availability of different host factors and the ability to readily assemble viral replication organelles (VROs). Genome-wide screens and proteomics approaches with Tomato Bushy Stunt Virus (TBSV) in a yeast model host indicated that tombusviruses rely on the cellular cytoskeleton to reorganize the cellular environment of their hosts. Using temperature-sensitive (ts) mutants of beta and gamma-tubulin proteins and pharmacological inhibitors, I demonstrated that the dynamic microtubular network restricts TBSV replication. Moreover, changes in the structure of microtubules greatly interfere with the actin structure as well, leading to problems in the subversion of selected host factors into replicase complexes and the enrichment of sterols at replication sites. In addition to the efficient recruitment of co-opted host factors, lipids and metabolites to the sites of viral replication, tombusviruses promote the biogenesis and accumulation of host factors that facilitate the production of energy required to fuel replication. I discovered that Centromeric Histone H3 (CENH3), an essential chromatin-associated protein, has a non-canonical role during virus replication, as a regulator of the biosynthesis of several glycolytic enzymes that are necessary to generate ATP within the viral replication compartment. This function is achieved by the binding of this protein with components of the viral replication machinery such as the RNA chaperone p33 and the viral repRNA, a function that is initially inhibitory but that is circumvented by the virus to reach optimal replication. Altogether, the studies with the microtubule cytoskeleton and CENH3 revealed an emerging picture for (+)RNA tombusviruses, suggesting that the extensive rewiring of metabolic pathways and remodeling of cellular membranes that support viral replication, requires the activities of particular kinds of cell-intrinsic restriction factors (CIRFs). These types of factors, which I called negative regulatory CIRFs, have an intrinsic inhibitory function but are exploited by the virus to achieve robust replication at the expense of certain viral resources

Modulation of transcription and alternative splicing by spliceosome-activating nineteen complex (NTC) in Arabidopsis thaliana

Components of the spliceosome activating NTC (Nineteen Complex) are conserved in eukaryotes and their mutations in mammals result in early embryonic lethality. In Arabidopsis, the core PRL (PLEIOTROPIC REGULATORY LOCUS) NTC subunit is encoded by two closely related genes PRL1 and PRL2. This provides the unique opportunity to study the regulatory roles of NTC using mutations of the PRL homologs. Whereas inactivation of PRL1 results in dwarfism, early flowering, altered leaf development and hypersensitivity to low temperature, sucrose, ABA and cytokinin, the prl2 mutations cause embryo lethality at the heart stage. Compared to PRL1, the PRL2 mRNA level is about 10-fold lower in both wild type and prl1 mutant seedlings. The viability of prl1 mutant is thus likely maintained by partially overlapping function of PRL2, which secures reduced but still sufficient level of spliceosome activation. Main aim of this work was to characterize the roles of PRL1 and PRL2 in the regulation of transcription and alternative splicing. Deep RNA-Seq analysis identified 5,814 differential expression (DE) and alternative splicing (DAS) events indicating 5-fold higher representation of unspliced introns in upregulated versus downregulated mRNA isoforms in the prl1 mutant compared to wt. About 25% of DAS events identified altered splice sites, and 30% changed mRNA’s ORF removing either N-terminal organellar or secretion signal peptides, or increasing C-terminal distance between polyA and translational stop codons to enhance mRNA degradation by nonsense-mediated decay (NMD). 25% of DE mRNAs carried alternative 5’ or 3’ ends indicating that the prl1 mutation also influences the positions of transcription start and polyadenylation/cleavage. Functional reannotation and GO analysis of dataset indicated upregulation of genes involved in cell division, UV response, DNA repair, iron uptake, anthocyanin biosynthesis, and ABA/oxidative stress responses, in contrast to downregulation of genes in cell elongation, vesicular transport, auxin signaling and redox-regulated pathogen defense and cell death pathways correlating with known phenotypic traits of the prl1 mutant. In a subset of DE genes, we characterized the effect of prl1 mutation on alternative splicing of ribosomal protein genes. To obtain prl2 mutant cell populations, we used a conditional genetic complementation system for generation of genetic mosaics, in which the prl2 mutant sectors were labelled by expression of the green fluorescent protein (GFP). Following enrichment of mosaic sectors in callus and cell suspension cultures, the GFP+ prl2 and GFP- wild type cell populations were separated by cell sorting and subjected to a similar deep RNA-Seq analysis. This study identified 6314 DE/DAS events, which were compared to those observed in prl1. This lead to the identification of numerous candidate genes implicated in meiotic recombination, fertilization, embryogenesis, DNA replication and post-embryonic development which are differentially downregulated in the prl2 mutant. These data suggest that NTC complexes carrying PRL1 and PRL2 control transcription and splicing of mRNAs coding for both overlapping and distinct cellular functions

Studies of cytoplasmically inherited genes for components of the mitochondrial ATP ase complex: analysis of the Oli-2 region of the mitrochondrial genome of 'Saccharomyces cerevisiae'

This thesis describes studies of the structure and organisation of the Oli-2 region of the mitochondrial genome from wild type, drug resistant (01i2 . Ossl ) and mit strains of the yeast Saccharomyces cercvisiac . In addition attempts have been made to identify the locus of cytoplasmic, nonmitochondrial markers (VENR , TET R, Rh8GR), to drugs which affect mitochondrial energy metabolism. Firstly, a fine structure genetic map of the Oli-2 region of the mitochondrial genome has been generated by petite deletion mapping. Two mit mutations (pho8 and pho9) have been mapped upstream and one mit mutation (mit-175) has been mapped downstream of the 01i2 locus (Chapter-2). A problem was identified regarding the effect of a nuclear gene ( kar-1) on the copy number and transmission of petite (p ) mitochondrial genomes I Chapter-3). To circumvent this problem the relevent portions of the mitochondrial DNA l mt-DNA) from various mutants of the Oli-2 region have been cloned in a multicopy plasmid. pAT 153 of E. colI, for their amplification and propagation (Chapter-4). The cloned DMAs have been sequenced using the single stranded phage, M 13 as a sequencing vector. A sequence of about 4000 bp. starting from the Carboxyl terminal end of cytochrome oxidase subunit-1 (Oxi-3) to 1500 bp downstream of 01i2 has been sequenced. Three main reading frames have been identified in this stretch of the DNA segment. The mutations (0/i'2 , Oa 1 ) leading to the resistivity towards the drugs oli- gomycin and ossamycin have been located in the reading frame for subunit-6. The mit mutation (pho9) has been found not to lie on the structural gene for subunit-8 which is located upstream of the gene for subunit-6. It is assumed that the mutation is possibly located in the intergenic regulatory region of the genes. A putative reading frame has been identified downstream of subunit-6 reading frame, which could be the possible site for the genetic locus, mit-175 (Chapter-5). Models for the secondary and tertiary structures of subunit-6, subunit-8, and subunit-9 have been proposed on a theoretical basis using hydrophobicity plots and a modified Chou and Fasman method (Chou & Fasman, 1978). Suggestions as to the mechanism of inhibi­tion of oxidative phosphorylation by oligomycin and ossamycin have been made on the basis of these models. The present studies also indicate that subunit-8 has structural analogies to subunit-b of E. colt ATP synthetase (Chapter-6). An attempt has also been made to identify a cytoplasmic candidate for nonmitochondrial, cytoplasmic genetic markers (P£,VR, TETR, Rh6GR) with special emphasis on studies of the 3 μ plasmid (Chapter-7). It has been demonstrated that the 2μ plasmid, or dsRNAs found in the cytoplasm are not the bearer of these genetic markers. In spite of the fact that the 3 μ DNA species under investigation has an insertion, no evidence has been obtained that the 3 μ plasmid contain the above markers. The existence of a high molec­ular weight plasmid in the cytoplasm of S. cercvisiae has also been demonstrated