Résumé :
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Spinal muscular atrophy is a neuromuscular disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN is part of a macromolecular ("SMN")-complex that, together with the PRMT5-complex mediates the assembly of Sm proteins onto spliceosomal U snRNPs. We have defined different steps of the assembly pathway in an in vitro assay that closely mimics in vivo conditions. After translation in the cytoplasm Sm proteins are initially sequestered by the PRMT5-complex. In a second step, the Sm proteins are directly transferred to the SMN-complex. The Sm protein transfer is dependent on the prior modification of arginines in B/B', D1 and D3 to symmetrical dimethylarginines, catalyzed by the methyltransferase PRMT5. In the last step of the assembly reaction, the Sm proteins are loaded onto the U snRNA. Finally, we have employed gene silencing to assess the effect of reduced levels of the SMN protein on UsnRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to pathological levels impairs UsnRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less stringent conditions, zebrafish embryos proceeded through development, yet exhibited dramatic SMA-like motoneuron degeneration. The same was observed after silencing two other essential factors in the UsnRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified UsnRNPs into SMN-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of UsnRNPs.
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