Résumé :
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Spinal muscular atrophy (SMA) is caused by mutations of Survival Motor Neuron 1 (SMN1) gene, which lead to reduction of SMN protein levels. The ubiquitous SMN complex participates to the biogenesis of spliceosomal core snRNPs and together are enriched into nuclear gems/Cajal Bodies (CBs). There is a tight correlation between the number of gems/CBs and the severity of SMA disease. Moreover, gems/CBs from SMA-derived fibroblast cells show no detectable accumulation of snRNPs. The mechanism by which the SMN complex and snRNPs are associated/dissociated from gems/CBs remains elusive. It has been shown in recent studies that phosphorylation might regulate the function and localization of the SMN complex. Here, protein complexes were immunoprecipitated from COS cells stably expressing eGFP-tagged SMN mutants using anti-GFP antibodies. Silver staining of the coeluted proteins separated by SDS-PAGE revealed a band of about 42-kDa that was analysed by mass spectroscopy. A nuclear serine/threonine protein phosphatase was identified on the basis of 32 peptides covering 68% of its sequence. This enzyme might be critical for the biochemical properties of the SMN protein. We have characterized the association of this protein phosphastase with the SMN protein using immunoprecipitation experiments, siRNA-mediated depletion, 2D-PAGE and immunofluorescence analyses. These findings will be reported. Our proteomics-based strategy is to decipher the molecular functions of the SMN protein in gems/CBs, to improve our understanding of the pathological processes and hopefully, help clinical pratice.
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