Abstract EN:

Mitochondria are organelles found in nearly all eukaryotes. They are considered to be the energetic center of the cell because they generate ATP by oxidative phosphorylation, but they are also involved in many more biological processes such as lipid and amino acid metabolism, iron-sulphur (FeS) cluster biogenesis, calcium homeostasis and apoptosis. Mitochondria originate from the endosymbiosis of an alpha-proteobacterium ancestor into a proto-eukaryotic cell. Classical mitochondria have retained a highly reduced vestige of the genome of the ancestral bacteria such that most mitochondrial proteins but also numerous tRNAs have to be imported from the cytosol to the mitochondria (Sieber et al. , 2011a). Mitochondrial genomes are subject to numerous mutations that can result in mitochondrial dysfunctions, which are often dramatic for cell viability. Such mitochondrial disorders can be at the center of human neurodegenerative and neuromuscular diseases, diabetes, aging and also cancers (Florentz et al. , 2003). In plants, mitochondrial disorders can originate from the presence of chimeric sequences in mitochondrial genomes, which lead to cytoplasmic male sterility (CMS). CMS plants are incapable of producing functional pollen and constitute a valuable tool in agronomy to produce hybrid plants that are more vigorous in culture (Budar and Pelletier, 2001). Mitochondrial transformation is thus of great interest, both for the study of mitochondrial disorders, and as a biotechnological tool for example in agronomy. Mitochondrial gene expression also remains poorly characterized and awaits reverse genetics tools for a better understanding. Except for the yeast S. Cerevisiae and the unicellular algae C. Reinhardtii where stable mitochondrial transformation has been achieved by biolistic approaches (Fox et al. , 1988; Remacle et al. , 2006), no means exist to stably transform mitochondrial DNA of higher eukaryotes. In my Ph. D. , I focused on developing a tool for efficient introduction of exogenous RNA into mitochondria of various model organisms. The strategy was to use a nucleic acid binding protein fused to a mitochondrial targeting sequence to create a protein shuttle capable of targeting RNA substrates to the mitochondrial matrix. As a shuttle candidate, I chose the mouse dihydrofolate reductase (DHFR) that binds nucleic acids non-specifically in vitro. A mitochondrial targeting sequence fused to the protein allows it to be imported into isolated mitochondria. DHFR fused to a mitochondrial targeting sequence (pDHFR) is conventionally used to dissect the mechanism of protein import into mitochondria of yeast (Pfanner et al. , 1987), and was used as a starting point for this study.