Objective
An important number of human neurodegenrative diseases are associated with point mutations in mitochondrial DNA coded mitochondrial tRNAs. The long-term objective of this project is to study the possibility to complement these mutations by heterologous tRNAs expressed in the nucleus and then imported into the mitochondria. Two of the applicants have demonstrated that in the yeast S. cerevisiae, one cytoplasmic tRNA-Lys is selectively associated with the inner mitochondrial compartment.
In vitro and in vivo experiments have demonstrated that: this import requires energized mitochondria conserving the electrochemical potential across the mitochondrial inner membrane; it implies formation of a RNP compex on the organellar surface; it depends of the integrity of the mitochondrial pre-protein import machinery; aminoacylation of the tRNA by the cytoplasmic lysyl-tRNA synthetase KRS is prerequisite for import; the prrsor of the mitochondrial lysyl-tRNA synthetase pre-MSK is the essential factor of import. These data suggest that the tRNA is imported via the pre-protein import channel in a complex with pre-MSK. Finally recent data obtained by the applicants are clearly in favor of a possibility (i) to import thoroughly mutated tRNALys versions in yeast mitochondria and (ii) the tRNA-Lys can be imported in isolated human mitochondria in the presence of yeast import directing proteins.Development of an artificial tRNA import system for human cells will be based on our knowledge of the mechanisms of tRNA import in yeast. Such a system would permit to serve a basis for a development of novel gene therapy approach for several mitochondrial myopathies. To approach this objective, the following steps will be necessary:
To detemine import signals in the tRNA and to create on this basis new artificial importable strates.
To identify the mode and mechanism of interaction between the imported RNA and import directing proteins.
To develop a system of tRNA import in isolated human mitochondria by using, if necessary, import directing factors of yeast.
To assess that the imported tRNA can participate in organellar protein Synthesis.
To demonstrate tRNA import in model human cell lines by co- expressing genes coding for the tRNA and for heterologous factors essential for its import in vitro.
To test if the import of tRNA can complement a mitochondrial DNA mutation in a tRNA gene.
At long term, the objective of this project will be to define experimental conditions for gene therapy of mitochondrial diseases associated with mutation in tRNA genes, taking as a model of study the MERRF syndrome (resulting of a mutation in the mitochondrial tRNALys gene). The proposed work is quite innovative and, being a fundamental research, has clear applicable long-terms objectives. The research plan is stricktly based on the competences of the four participants and, on the other hand, the areas of experience of each team are complementary, including such fields as mitochondrial biochemistry and pathology in humans, yeast mitochondrial mollar biology and genetics and structural analysis of animal tRNAs and aminoacyl-tRNA synthetases. The project is placed in the frame of research activities already performed in the laboratories of the applicants. Financial support of these activities would permit to obtain significant progress in the field and to organize a network able to resolve problems which are not accessible for each applicant separately.
Topic(s)
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67084 Strasbourg
France