RNA import into mitochondria: targeting and translocation mechanisms – RNA-mitotar

Mitochondria are the organelles essential for functioning of the eukaryotic cell, which contain their own genome (mtDNA) and participate in a variety of processes, ranging from respiration-driven ATP synthesis to apopotosis. mtDNA contains only a limited genetic information, while the most of macromolecules are encoded in the nucleus and targeted in the mitochondria. In contrast with the protein import studied in details, RNA uptake by mitochondria is much less understood, in spite of a wide presence of this pathway among species. Since RNA import is a unique pathway of nucleic acids targeting into the mitochondria, investigation of its mechanisms is fundamental. On the other hand, it must be outlined that many human diseases are caused by mutations in mtDNA and that RNA import offers a possibility to complement these mutations. This possibility is currently in investigation in our team, in collaboration with the clinicists. Nevertheless, comprehension of the fine mechanisms of RNA import is far insufficient to respond to all the challenges raised by its biomedical exploitation. The current project aims to resolve this insufficiency. We propose to use two models of study, the yeast (S. cerevisiae) importing transfer RNAs (tRNAs) and human cells, which import 5S ribosomal RNA (5S rRNA) into mitochondria. These two models have already been exploited by our team to obtain several crucial data on RNA import, which were published in top-rate journals (Science, Genes & Dev, Mol Cell, EMBO J, PNAS, etc.). In yeast, proteins essential for tRNA import and RNA motifs determining the import specificity were identified and the mitochondrial function of the imported RNA was assigned. In human cells, the resemblance of 5S rRNA import conditions and those of tRNA import in yeast was observed and the sequences essential for 5S rRNA import were localized. The current project aims to extend these results and, by series of additional experiments, to permit integration of the RNA import mechanism in the complex pathways "patchwork" of the macromolecular traffic. The project comprises three main objectives: (1) identification of protein factors of RNA targeting and intra-mitochondrial translocation: (2) description of the molecular mechanisms of RNA-protein interaction between imported RNAs and import factors; (3) description of the structure-function relation for the two models studied. Targeting factors will be searched in human cells (those of yeast being already identified). Biochemical approaches (separation of macromolecules and in vitro import assays) coupled with proteomic methods will be used to seek for the candidates, followed by the reconstitution of the import system from recombinant molecules and final validation in vivo by RNA interference driven extinction of the corresponding genes. Mitochondrial receptors involved in RNA import will be addressed by masking of known surface receptors and RNA-protein cross-linking assays during import course. Interaction of the known and newly identified import factors with imported RNAs will be characterized by footprinting (to identify RNA-protein contacts) and gel-shift/native gels assays (to describe the dynamics of complex formation). The "functional" part of the project will be axed at two different objectives depending on the model. In yeast, the function of one imported tRNA was recently identified as adaptation of mitochondrial translation to stress conditions, which suggest the existence of conditional regulation. Indeed, implication of the Ubiquitine-Proteasome system (UPS) in tRNA import modulation was then discovered. We propose to identify, by genetic and biochemical approaches, the targets of the UPS in the tRNA import pathway. In human cells, the mitochondrial function of 5S rRNA remains unclear. To address it, we shall exploit our recent data on its import determinants. The motifs and bases known to interact with cytoplasmic or nuclear proteins (TFIIIa, RPL5, etc.) will be mutated without affecting import determinants and their effect on intracellular distribution of 5S rRNA will be studied. We also shall investigate if the imported 5S rRNA is associated with mitochondrial ribosomes. Finally, 5S rRNA import will be inhibited by siRNAs driving extinction of the genes expressing the identified import factors and the effect on various mitochondrial functions will be studied. The current project is strictly based on the know-how of our team in this particular field of research. Furthermore, taking into account numerous important pre-acquired data, I believe that it is feasible in its totality in the frame of 48 months.