Résumé :
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Introduction : Myotonic dystrophy type 1 (DM1) is a dominant neuromuscular disorder affecting 1/8000 individuals worldwide. This disease is caused by an unstable expansion of CTG trinucleotide repeats located in the 3' untranslated region of the dystrophy myotonic protein kinase (DMPK) gene (Fu et al, 1992). In muscle cells, the mutated DMPK transcript is retained in nuclear foci (Davis et al, 1997) where it sequesters the splicing factor MBNL1 (Mankodi et al, 2001). In addition, the activation of the protein kinase C in DM1 muscle cells leads to an increased stability of the splicing factor CUGBP1 (Kuyumcu-Martinez et al, 2007). Aim: Set up and phenotypically charactarize a new DM1 drosophila line to dissect the effect of time and the role played by each factor (Mbl and/or Bru-3) in larval somatic muscles of DM1 flies. Later, this line can be used for transcriptomic analyses. Method : Thanks to site-specific transgenesis, we generated a Drosophila model of DM1 expressing non-coding RNAs with 240, 480, 600 or 960 CUG interrupted repeats. In order to better understand the role played by MBNL1 and CUGBP1 in this disease, 2 lines were tested in parallel with DM1 lines: (i) bru-3 gain-of-function line that mimics CUGBP1 stabilization in muscle cells of DM1 patients (ii) line with attenuated mbl expression that mimics the decreased availability of MBNL1 in muscle cells of DM1 patients. Immunostainings associated with confocal microscopy allowed us to describe in vivo the effects of triplet repeat expression on muscle fibres and to attempt rescue experiments with a bru-3 deficiency line. Results : When non coding RNAs carrying CUG repeats were specifically expressed in somatic muscles, foci can be detected by in situ hybridization in all nuclei of muscle fibres of 3rd instar larvae and colocalize with the MBNL1 drosophila orthologue, Mbl. Antibody stainings confirmed both cytoplasmic and nuclear expression of Mbl and Bru-3 in larval and adult somatic and cardiac muscles. Larval motility is affected and associated with fibre splitting, reduced fibre size, affected nuclei fusion in all pathological lines. Besides, DM1960 and Mbl RNAi lines display affected fibre contractility and hypercontraction. Discusion - Conclusion : Both Mbl and Bru-3 factors seem to play a role in larval muscle phenotypes with Mbl sequestration being specifically involved in contraction and relaxation phenotypes. In silico genome wide analysis was performed to identify potential targets of Mbl and Bru-3. Candidate genes can now be tested in rescue experiments.
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