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

成年哺乳动物骨骼肌是由多个单核成肌细胞融合而成的高度定向的致密纤维束组成的稳定组织。然而，一旦受伤，修复过程，称为肌生成，立即启动，其中受损的纤维被替换，以防止肌肉质量的损失。肌生成需要肌生成调节因子（MRF）如肌生成素的表达。多年来，我们的研究计划旨在确定转录后机制在编码某些MRF的基因表达中的意义。我们已经表明，RNA结合蛋白（RBP）HuR，一个主要的转录后调节因子，是肌肉细胞分化所必需的。HuR通过与位于肌细胞生成素mRNA的3 '非翻译区（3' UTR）的富含AU的元件（战神）结合，导致其稳定化，从而促进肌细胞生成。我们的努力，以确定新的HuR mRNA的目标发现，HuR也促进肌生成的差异调节编码细胞因子HMGB 1和细胞周期调节剂核磷蛋白（NPM）的mRNA的表达。** 最近，HuR的功能在巨噬细胞中被证明是由一种新的翻译后修饰来调节的。这种修饰涉及通过聚ADP-核糖聚合酶1（PARP 1）对HuR进行聚ADP-核糖基化（PAR）。 PARP 1对HuR的PAR化增加了HuR与其mRNA靶点的结合，从而增加了这些信息的稳定性。 通过进行体外PAR化实验获得的我们的初步数据表明，除了PARP 1，HuR也可以被包括PARP 2、5a和5 b在内的其他PARPs PAR化。 有趣的是，我们的结果表明，只有PARP 5a在生肌过程中起作用。 事实上，PARP 5a而不是其他PARPs的敲低阻止了肌生成，这与HuR mRNA靶点NPM、肌生成素和HMGB 1的表达变化相关。我们的初步数据还表明，这种效应可能是由于HuR的PAR化减少，从而导致HuR与这些mRNA的结合减少。 尽管我们的数据清楚地表明PARP 5a介导的PAR化事件对肌生成过程的影响，但它们对HuR介导的转录后机制的调节的贡献仍然难以捉摸。 因此，这项发现基金将研究HuR的PAR化是否以及如何参与调节肌生成。 在这项资助中，我们将：**1）表征HUR中PARP 5a介导的PAR化位点，并评估它们在肌生成过程中的功能相关性 *2）确定PARP 5a介导的HUR的PAR化如何在转录后影响肌生成过程中NPM，肌生成素和HMGB 1 mRNA的表达 ** 通过确定PARP 5a对HUR介导的转录后事件及其在肌生成过程中的作用模式的影响，我们的工作将有助于发现对肌肉形成重要的新的调控过程。

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.********************