The role of post-translational modifications in miRISC function

 描述(申请人提供)：由microRNAs介导的转录后基因沉默(MiRNAs)是后生动物基因调控的一个既定范例，在包括发育时机、器官发生、细胞分化和增殖在内的一系列生物学过程中发挥关键作用。调控miRNA生物发生和靶向沉默的机制在很大程度上已经建立；然而，我们对miRNA介导的沉默复合体(MiRISC)的翻译后修饰如何影响其稳定性和完整性、亚细胞分布以及抑制靶基因表达的效应器功能的了解仍然存在重大缺陷。我们的长期目标是了解miRISC的翻译后修饰如何影响miRNA介导的基因沉默。这项建议的具体目的是通过对miRISC核心亚基的磷酸化来研究蛋白激酶CK2在miRNA途径中的新作用。我们的初步数据表明，CK2通过两个miRISC组分的磷酸化作用与miRISC相互作用：死盒解旋酶CGH-1和血管内含子基因产物VIG-1。根据我们的初步数据，我们假设CK2通过CGH-1和VIG-1的磷酸化在促进miRISC与靶mRNAs结合方面发挥关键作用。我们将通过追求两个特定的目标来验证我们的假设：1)评估CGH-1和VIG-1磷酸化在miRNA靶标结合和表达的特异性中的关键作用及其在动物发育过程中的生物学意义。2)明确CK2活性和miRISC磷酸化对miRISC组成完整性的作用及其对miRNA靶降解中心P小体的募集。此外，我们还将探索候选PP2A磷酸酶对miRISC磷酸化状态的相反作用对CK2活性的潜在调节作用。这项研究具有重要意义，因为它将建立miRISC的翻译后修饰作为miRISC组装、定位和沉默功能所需的一般机制，从而影响由miRNA介导的基因沉默调控的所有生物途径。所获得的基本见解也将为治疗复杂人类疾病的快速发展的miRNA疗法领域提供信息。

 DESCRIPTION (provided by applicant): Post-transcriptional gene silencing mediated by microRNAs (miRNAs) is an established paradigm in metazoan gene regulation, playing critical roles in a wide array of biological processes including developmental timing, organogenesis, and cellular differentiation and proliferation. Mechanisms governing miRNA biogenesis and target silencing are largely established; however, a significant deficit remains in our understanding of how post- translational modifications of the miRNA-mediated silencing complex (miRISC) affect its stability and integrity, subcellular distribution, and effector functio in repressing target mRNA expression. Our long-term goal is to understand how post-translational modifications of miRISC influence miRNA-mediated gene silencing. The specific objective of this proposal is to investigate a novel role for protein kinase CK2 in the miRNA pathway through phosphorylation of core miRISC subunits. Our preliminary data indicate that CK2 physically interacts with miRISC through phosphorylation of two miRISC components: the DEAD-box helicase CGH-1 and the vasa intronic gene product VIG-1. Based on our preliminary data, we hypothesize that through phosphorylation of CGH-1 and VIG-1, CK2 plays a critical role in promoting miRISC binding to target mRNAs. We will test our hypothesis by pursuing two specific aims: 1) Evaluate the key role of CGH-1 and VIG-1 phosphorylation in the specificity of miRNA target binding and expression and their biological significance during animal development. 2) Define the role of CK2 activity and miRISC phosphorylation on the compositional integrity of miRISC and its recruitment to the miRNA target degradation centers, the P bodies. In addition, we will explore potential regulation of CK2 activity by the opposing function of candidate PP2A phosphatases on the phosphorylation state of miRISC. The proposed research is significant because it will establish post-translational modification of miRISC as a general mechanism required for miRISC assembly, localization, and silencing function that impacts all biological pathways regulated by miRNA-mediated gene silencing. The basic insights obtained will also inform the rapidly advancing field of miRNA therapeutics for treating complex human diseases.