Résumé :
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DMD is caused by mutations/deletions in the X-linked dystrophin gene. Several studies have shown that the dystrophin homologue, utrophin, can functionally compensate for the lack of dystrophin in muscle when expressed at appropriate levels and at the correct subcellular location. Thus, it becomes important to identify the mechanisms regulating utrophin in attempts to increase its expression in dystrophic muscle. Previous work from our lab established that in addition to transcriptional control, expression of utrophin is also tightly regulated by post-transcriptional events. In this study, we specifically examined the role of AU-rich elements and the mRNA-binding protein KSRP, on utrophin RNA stability. Using luciferase reporter constructs containing the utrophin 3'UTR, we show that shRNA-mediated KSRP knockdown results in a two-fold increase in luciferase activity. By contrast, overexpression of KSRP in N2A cells induced a 70% decrease in luciferase activity. Furthermore, overexpression of KSRP led to a significant decrease in endogenous utrophin mRNA and protein expression whereas a threefold increase in endogenous utrophin A transcript and protein was seen when KSRP is suppressed. To demonstrate an association between KSRP and utrophin mRNAs, TA muscle extracts and C2C12 cell lysates were crosslinked and immunoprecipitated with either anti-KSRP antibody or IgG. qRT-PCR analysis revealed that utrophin mRNAs are indeed associated with KSRP in muscle cells. In vivo studies revealed that electroporation of mouse TA muscles with both sh-KSRP and utrophin 3'UTR luciferase constructs resulted in a significant increase in luciferase activity after 7 days. This increase in reporter activity was accompanied by an increase in endogenous utrophin A expression. Taken together, these data clearly demonstrate that KSRP regulates the abundance of utrophin mRNA via post-transcriptional events and provide key insights into possible therapeutic interventions aimed at increasing utrophin levels in dystrophic muscle. Funded by AFM, CIHR and MDA (USA).
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