Biogenesis and Function of the Mitochondrial Ribosome
Repository URI
Repository DOI
Change log
Authors
Abstract
In this thesis, I present results of my PhD research into the biogenesis and function of the human mitochondrial ribosome. The mammalian mitochondrial ribosome (mitoribosome) is one of the largest ribonucleoprotein complexes in the cell, with the overall molecular mass of the fully assembled 55S monosome being ~2.7 MDa. It is indispensable for cell survival, as it translates 13 polypeptides encoded in mitochondrial DNA, which are essential for oxidative phosphorylation and therefore for supplying the cell with energy in the form of ATP. The mammalian mitoribosome consists of small 28S and large 39S subunits. It is of dual genetic origin, with all protein components encoded in the nucleus and all RNA components encoded in the mitochondrial DNA. A total of 82 proteins, 2 rRNAs and a structural tRNA comprise one mitoribosomal particle. Proper function and assembly of this molecular machine is reliant on numerous associated factors, such as RNA modifying enzymes and assembly factors. Although recent significant progress has been made in our understanding of how the mitoribosome assembles and its functions in translation, we still lack the knowledge about many factors that are necessary for these processes. This work aims to provide insight into the function of selected proteins that were predicted to be involved in biogenesis and/or function of mitochondrial ribosome, namely mitochondrial rRNA methyltransferase 1 (MRM1) and GTP binding protein 8 (GTPBP8). The work also presents genomics and proteomics methods for the study of mitochondrial gene expression machinery and of mitoribosome integrity and assembly, respectively. The first two chapters provide background information for the research performed in the remaining part of the thesis. In the first chapter, I summarise current knowledge of mitochondrial gene expression, with a focus on post-transcriptional RNA modifications and mitochondrial translation. The second chapter describes the materials and methods used. In the third chapter, I present a computational method for analysis of complexome profiling data from experiments that employ stable isotope labelling by amino acids in cell culture (SILAC). This method is implemented in R and is freely available as the Bioconductor package ComPrAn. It provides tools for analysis of peptide-level data as well as normalisation and clustering tools for protein-level data, dedicated visualisation functions and is accompanied by a graphical user interface. Throughout this thesis ComPrAn has been used for quantitative and qualitative analysis of mitoribosomes in studied cell lines. In the fourth chapter, I introduce a CRISPR/Cas9-based screening approach designed to target genes with known or predicted function in mitochondrial gene maintenance and expression. I apply this method to identify genes that show genetic interaction with MRM1. The screen identifies MRM2 as a top candidate for genetic interaction with MRM1. This finding is followed up by the generation of a double knockout cell line which shows severe mitochondrial deficiency, with uridine auxotrophy and disruption of assembly of small mitoribosomal subunit being the most striking effects observed. These findings provide further insight into the role of MRM1 in mitochondria and highlights the complexity of regulation of mitochondrial translation. The fifth chapter focuses on establishing the role of uncharacterised GTPBP8 protein in the cell. I localised GTPBP8 to mitochondria and studied its function by production of a knockout cell line. GTPBP8 knockout presents a strong oxidative phosphorylation defect due to impaired mitochondrial translation. Quantitative analysis of mitoribosome reveals accumulation of both small and large subunits in the knockout, suggesting that GTPBP8 might play a role in very late assembly of either of the subunits, subunit joining or translation initiation. Overall, this work improves our understanding of the regulation of mitochondrial translation by characterising two mitochondrial proteins and their role in mitoribosome biogenesis and function. The CRIPSR/Cas9 screening methodology and ComPrAn R package presented here have potential to be used in the study of other proteins, extending the portfolio of methods available for research of mitochondrial function.
Description
Date
Advisors
Keywords
Qualification
Awarding Institution
Rights
Sponsorship
European Commission Horizon 2020 (H2020) (721757)