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

翻译后修饰对肌纤维形成过程中 RNA 结合蛋白功能的作用

• 批准号: RGPIN-2019-06111
• 负责人: Gallouzi, Imed
• 金额: $ 2.33万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684464
• 关键词: role; posttranslational; modifications; function; RNA

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

哺乳动物成年骨骼肌是一种稳定的组织，由几束高度定向的致密纤维组成，来源于几个单核成肌细胞的融合。然而，一旦受伤，一个被称为肌生成的修复过程就会立即启动，在这个过程中，受损的纤维被替换，以防止肌肉质量的损失。肌生成需要表达肌生成调节因子（MRFs），如肌生成素。多年来，我们的研究计划旨在确定编码一些mrf的基因表达的转录后机制的含义。我们已经证明，rna结合蛋白（RBP） HuR，主要的转录后调节因子之一，是肌肉细胞分化所必需的。HuR通过与位于肌生成素mRNA 3‘非翻译区（3’UTR）的富au元件（AREs）结合促进肌生成，导致其稳定。我们努力鉴定新的HuR mRNA靶点，发现HuR还通过差异调节编码细胞因子HMGB1和细胞周期调节剂核磷蛋白（NPM）的mRNA的表达来促进肌肉形成。******最近，巨噬细胞中HuR的功能被一种新的翻译后修饰所调节。这种修饰涉及到多adp核糖聚合酶1 （PARP1）对HuR的多adp核糖基化（PAR）。PARP1对HuR的PARylation增加了HuR与其靶mRNA的结合，从而增加了这些信息的稳定性。我们通过体外PARylation实验获得的初步数据表明，除了PARP1之外，HuR还可以被PARPs 2、5a和5b等其他PARPs所parp化。有趣的是，我们的结果表明，只有PARP5a在肌生成过程中起作用。事实上，敲低PARP5a而不敲低其他PARPs可以阻止肌肉发生，这与HuR mRNA靶蛋白NPM、myogenin和HMGB1的表达变化有关。我们的初步数据还表明，这种影响可能是由于HuR的PARylation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证明了PARP5a介导的PARylation事件对肌生成过程的影响，但它们对hr介导的转录后机制的调节作用仍然难以捉摸。因此，这项发现将研究HuR的PARylation是否以及如何参与调节肌肉形成。在这项资助中，我们将：******1)表征HuR中PARP5a介导的PARylation位点，并评估其在肌肉发生过程中的功能相关性***2)确定PARP5a介导的HuR的PARylation转录后如何影响肌肉发生过程中NPM、肌生成素和HMGB1 mrna的表达******通过确定PARP5a对hr介导的转录后事件及其在肌肉发生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节过程。********************