Messenger RNA Turnover in Mammalian Cells

描述（由申请方提供）：细胞质mRNA的降解在基因表达的调节和mRNA生物发生的质量控制中起着重要作用。几乎所有的真核生物mRNA降解途径都是由去腺苷化启动的，即，3' poly（A）尾的缩短，在哺乳动物细胞中有两个不同的阶段。去腺苷化的一个主要结果是形成不可翻译的mRNA-蛋白质复合物（mRNP），其导致mRNA衰变或翻译沉默。去腺苷酸化通常是mRNA衰变和翻译沉默的限速步骤，使其成为控制胞质mRNA功能和命运的重要点。然而，目前对哺乳动物细胞中如何调节这一基本过程知之甚少。特别是，目前还不清楚在去腺苷化的两个阶段中的每一个阶段中发生了什么样的mRNP重塑，以及单个mRNP重塑步骤通常如何影响细胞质mRNA的命运。在过去的资助期间，我们已经取得了几项关键发现，为解决哺乳动物细胞中的这些重要问题奠定了基础。我们发现：1）miRISC复合物通过募集Pan 2-Pan 3和Ccr 4-Caf 1去腺苷酶复合物来分别加速靶mRNA去腺苷化的第一和第二阶段，从而触发快速mRNA衰变; 2）去腺苷化的第一阶段由Pan 3的两种功能不同的同种型协调，Pan 3是Pan 2去腺苷酶的调节因子; 3）TNRC 6的内在无序区（IDR）中的可逆磷酸化（一种微小mRNA（miRNA）介导的基因沉默的效应子）和Pan 3调节这些蛋白与细胞质多聚腺苷酸结合蛋白（PABP）C1和/或PABP C2的相互作用。或去腺苷酸酶，这反过来又控制它们的细胞功能;和4）TNF和ROCK激酶抑制剂分别对miRNA介导的基因沉默有负面和正面影响。在本研究中，我们将：1）确定Pan 3L和Pan 3S亚型在mRNA去腺苷化调节中的不同作用的基础以及Pan 3 IDR磷酸化在该过程中的作用; 2）阐明去腺苷化的双相性质的生物学意义以及在去腺苷化的每个阶段发生的mRNP重塑的细节;和3）阐明了通过调节miRISC复合物促进去腺苷化的能力来全面调节miRNA介导的mRNA衰变的新机制。拟议的研究将提供一个机制框架，了解动态和信号依赖性控制细胞质mRNA的功能，通过内在无序的蛋白质。它们也代表了对我们长期目标的有意义的进展，即理解哺乳动物细胞中mRNP背景下管理细胞质mRNA代谢的生物学原理和机制。

DESCRIPTION (provided by applicant): Degradation of cytoplasmic mRNA plays an essential role in the regulation of gene expression and in quality control of mRNA biogenesis. Nearly all eukaryotic mRNA decay pathways are initiated by deadenylation, i.e., the shortening of the 3' poly(A) tail, which has two distinct phases in mammalian cells. One major consequence of deadenylation is the formation of non-translatable mRNA-protein complexes (mRNPs), which leads to mRNA decay or translation silencing. Deadenylation is often a rate-limiting step for mRNA decay and translational silencing, making it an important point for controlling the function and fate of cytoplasmic mRNA. However, presently little is known about how this fundamental process is regulated in mammalian cells. Particularly, it is unclear what kind of mRNP remodeling occurs during each of the two phases of deadenylation and how individual mRNP remodeling steps impact the fate of cytoplasmic mRNA in general. In the past funding period, we have made several key findings that lay the groundwork for addressing these important questions in mammalian cells. We discovered that: 1) miRISC complex triggers rapid mRNA decay by recruiting both Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes to accelerate the first and second phases, respectively, of target mRNA deadenylation; 2) the first phase of deadenylation is coordinated by two functionally distinct isoforms of Pan3, a regulatory factor of Pan2 deadenylase; 3) reversible phosphorylation in intrinsically disordered regions (IDRs) of TNRC6 (an effector for micro mRNA (miRNA)-mediated gene silencing) and of Pan3 modulates the interactions of these proteins with cytoplasmic poly(A)-binding protein (PABP)C1 and/or deadenylases, which in turn controls their cellular functions; and 4) TNF¿ and ROCK kinase inhibitors have negative and positive effects, respectively, on miRNA-mediated gene silencing. In the current proposal, we will: 1) determine the basis for the distinct actions of the Pan3L and Pan3S isoforms in the regulation of mRNA deadenylation and the role of phosphorylation in the Pan3 IDRs in the process; 2) elucidate the biological significance of the biphasic nature of deadenylation and the details of mRNP remodeling that occur in each phase of deadenylation; and 3) elucidate novel mechanisms by which miRNA- mediated mRNA decay is regulated globally via modulating the ability of miRISC complexes to promote deadenylation. The proposed studies will provide a mechanistic framework for understanding dynamic and signal-dependent control of cytoplasmic mRNA functions via intrinsically disordered proteins. They also represent a meaningful advance towards our long-term goal of understanding the biological principles and mechanisms that govern cytoplasmic mRNA metabolism in the context of mRNPs in mammalian cells.