Developing tools to investigate initiating events of mitochondrial translation

Abstract

Mitochondria are the predominant source of adenosine triphosphate (ATP) in nucleated human cells. ATP is required as cellular energy source and mitochondria synthesise ATP by coupling the oxidative phosphorylation (OXPHOS) system. Mitochondria contain their own circular DNA (mtDNA) which encodes 11 messenger RNA (mt-mRNA), 2 ribosomal RNA (mtrRNA), and 22 tRNA (mt-tRNA) molecules which are all utilized for the synthesis of 13 proteins, all key components of the multi-subunit OXPHOS complexes, by the mitochondrial ribosome (mitoribosome). Much of the life cycle of mt-RNA and the process of translation is still unclear, in particular a full understanding of mt-mRNA stability, delivery, and loading onto the mt-SSU (or mitoribosomes) is lacking. The aim of this research was to investigate these processes using super resolution imaging of mt-rRNA and proximity-dependent proteomic analysis. I have visualised mt-rRNA molecules beyond the diffraction limit by the development and application of RNA Fluorescent In Situ Hybridisation (FISH) compatible with STimulated Emission Depletion (STED) microscopy. This technique was used to validate the loss of the 16S mt-rRNA upon induction of the mitochondrially-targeted VapC20 endonuclease and has contributed to a model wherein the formation of the monosome is an important factor in mtmRNA transcript stability. To further understand the process of mt-mRNA loading onto the mitoribosome, I employed proximity-dependent biotin identification (BioID) to uncover proteins which may be enriched at the mitoribosome mt-mRNA entry site. Multiple mitoribosomal proteins were fused to the BioID moiety, expressed in human cells, and screened for their ability to assemble into the mitoribosome. Mass spectrometry was used to characterise the proximal interaction network of MRPS39 and MRPS27 which uncovered the heterodimeric proteins PHB and PHB2. These proteins were then investigated to further understand their roles in mitochondrial translation. This thesis presents a thorough investigation into mt-RNA and mitochondrial translation and provides tools and techniques that complement the current literature.