The role of posttranslational modifications on the function of RNA binding proteins during muscle fiber formation

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Mammalian adult skeletal muscle is a stable tissue composed of bundles of highly oriented, dense fibers derived from the fusion of several mononucleated myoblasts. However, upon injury, a repair process, known as myogenesis, is immediately initiated in which damaged fibers are replaced to prevent the loss of muscle mass. Myogenesis requires the expression of myogenic regulatory factors (MRFs) such as myogenin. Our research program, over the years, has aimed to determine the implication of posttranscriptional mechanisms in the expression of genes encoding some of the MRFs. We have shown that the RNA-binding protein (RBP) HuR, one of the main posttranscriptional regulators, is required for muscle cell differentiation. HuR promotes myogenesis by associating with an AU-rich element (AREs) located in the 3'untranslated regions (3'UTR) of the myogenin mRNA, leading to its stabilization. Our effort to identify novel HuR mRNA targets uncovered that HuR also promotes myogenesis by differentially modulating the expression of mRNAs encoding the cytokine HMGB1 and the cell cycle modulator Nucleophosmin (NPM). ******Recently, the function of HuR was shown, in macrophages, to be modulated by a novel, posttranslational modification. This modification involves the poly-ADP-ribosylation (PAR) of HuR by poly-ADP-ribose polymerase 1 (PARP1). The PARylation of HuR by PARP1 increased the binding of HuR to its mRNA targets, resulting in the increased stability of these messages. Our preliminary data, obtained by performing in vitro PARylation experiments, suggests that in addition to PARP1, HuR can also be PARylated by additional PARPs including PARPs 2,5a and 5b. Interestingly, our results demonstrated that only PARP5a was however functional during the myogenic process. Indeed, knockdown of PARP5a but not the other PARPs prevented myogenesis which was correlated with changes in the expression of the HuR mRNA targets NPM, myogenin and HMGB1. Our preliminary data also show that this effect is likely due to the decreased PARylation of HuR, resulting, consequently, in the decreased binding of HuR with these mRNAs. Although our data clearly demonstrates the impact of PARP5a mediated PARylation events on the myogenic process, their contribution to the regulation of HuR-mediated posttranscriptional mechanisms remains elusive. This discovery grant, therefore, will investigate whether and how the PARylation of HuR is involved in regulating myogenesis. In this grant, we will:******1) Characterize the PARP5a-mediated PARylation sites in HuR and to assess their functional relevance during myogenesis***2) Determine how the PARP5a-mediated parylation of HuR posttranscriptionally affects the expression of the NPM, myogenin and HMGB1 mRNAs during myogenesis******By determining the impact of PARP5a on HuR-mediated posttranscriptional events and their mode of action during myogenesis, our work will help discover novel regulatory processes important for muscle formation.********************