Biomolecular Sciences

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Browsing Biomolecular Sciences by Subject "Alternative Spacing"

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    Expression, subcellular localization and functional characterization of RBM5 and RMB10 during the differentiation of C2C12 skeletal myoblasts (skeletal myogenesis)
    (2015-03-26) Masilamani, Twinkle Jasmine
    RNA-binding proteins (RBPs) are a highly regulated, evolutionarily conserved and functionally distinct family of proteins involved in key RNA metabolic processes. The RNA-binding motif protein RBM5 is an anti-proliferative, pro-apoptotic, putative tumor suppressor. A paralogue of RBM5, RBM10, which shares 50% identity with RBM5, functions in development. RBM5 and RBM10 are spliceosomal components involved in alternative splicing. RBM5 and RBM10 are ubiquitously expressed with higher levels in muscle (heart and skeletal) and pancreas. Most of the studies on RBM5 and RBM10 have been focused on cancer cells. Several factors such as 1) abundance in muscle, 2) developmental and temporal regulation, 3) alternative splicing activity and 4) association with functional events related to muscle development led us to hypothesize that both RBM5 and RBM10 are involved in skeletal muscle differentiation. The mechanism of action through which these two RBPs effect differentiation is hypothesized to involve alternative splicing of muscle differentiation-specific mRNAs. RBM5 and RBM10 expression and intracellular distribution was analyzed during muscle differentiation in the C2C12 murine model using qPCR, end-point PCR, immunoblotting and confocal microscopy. Also, RBM5 and RBM10 levels were transiently down-regulated using siRNA either separately and/or together and the associated changes in cell phenotype, expression of myogenic proteins plus a few alternative splicing events were analyzed. We observed a decrease in RBM5 and RBM10 protein expression levels in the differentiated myotubes compared to the myoblasts and myocytes, which indicates a time-dependent potential regulatory role during differentiation. Further, changes in RBM5 and RBM10 protein expression without modulating the levels of mRNA variants suggests posttranscriptional and/or post-translational regulation. Stage-specific differential localization suggests multiple functions related to mRNA biogenesis. RBM5-depleted cells showed a reduction in the total cell number during differentiation, and exhibited a delay in differentiation, fusion and maturation with down-regulated expression of myogenin and myosin heavy chain (MyHC). This implies that RBM5 is necessary to maintain the cell population to execute the myogenic differentiation process in a timely manner. RBM10- depleted cells showed an increase in total cell number immediately after transfection, and exhibited a delay in differentiation with a decrease in inclusion of exon 11 in Dtna mRNA. This indicates that RBM10 is required to maintain the necessary cell population before induction and acts as a splicing regulator during differentiation. RBM5- and RBM10- depleted cells differentiated and matured slowly, and had an increase in Mef2c γ exon inclusion. Therefore, these two RBPs are associated with the alternative splicing of Mef2cγ during differentiation. This is the first study to analyze the expression and the function of these two RBPs in a murine skeletal muscle differentiation model, and has implicated them in myogenesis, paving a way for further characterization. Future studies can investigate the involvement of RBM5 and RBM10 in disease states such as muscular dystrophy and rhabdomyosarcomas, given the known functions of RBPs in tumorigenesis in other cell types.