Investigating how mRNA decapping factors regulate gene silencing programs

转录后控制（PTC）程序在信使（m）RNA从其DNA模板转录后调节蛋白质产生。许多PTC程序通过抑制mRNA翻译和/或启动mRNA不稳定来控制蛋白质产生，这些事件统称为“沉默”。许多PTC介体，包括微（mi）RNA和RNA结合蛋白，通过募集CCR 4-NOT去腺苷酶复合物以靶向mRNA来引起基因沉默。其他的，如RNA结合蛋白MARF 1核糖核酸内切酶，内部切割靶向mRNA，使其衰变。miRNA机制和MARF 1都与mRNA去帽因子相互作用，这反过来又可以分别增强或抑制它们的活性。许多去盖因子通过多价相互作用网络相互关联。这些蛋白质-蛋白质相互作用网络可以反过来导致细胞中动态核糖核蛋白颗粒的形成，称为加工体或“P”体。这些P体及其蛋白质-蛋白质相互作用如何帮助调节mRNA生物学尚不清楚，但这是一个重要的研究课题。 在这里，我提出了一个研究计划，旨在阐明如何在mRNA去帽机制调节RNA颗粒完整性的背景下，不同类别的mRNA沉默程序。我们假设（i）mRNA去帽因子抑制MARF 1内切酶活性，（ii）调节RNA颗粒完整性反过来调节MARF 1和microRNA介导的基因沉默程序，以及那些由招募CCR 4-NOT去腺苷酶复合物的RNA结合蛋白介导的基因沉默程序。为了验证这些假设，我们提出了以下目标：1.阐明MARF 1如何与去盖机制相互作用的分子基础，以及它如何靶向选择的mRNA进行衰变。2.研究P体颗粒完整性对MARF 1介导的mRNA降解的调节作用。3.确定P体完整性如何调节microRNA抑制靶mRNA的方式。 拟议的研究计划是非常重要的，并建立在关键的已发表和未发表的意见，我们的小组已经在我们以前的NSERC发现赠款的过程中。最终，我们相信，这项研究提案将产生新的见解的模式和机制，通过mRNA脱帽和衰变因子之间的相互作用，帮助调节哺乳动物细胞中的PTC沉默程序。

Post-transcriptional control (PTC) programs regulate protein production after a messenger (m) RNA is transcribed from its DNA template. Many PTC programs control protein production by repressing mRNA translation and/or initiating mRNA destabilization, events collectively referred to as `silencing'. A number of PTC mediators, including micro (mi)RNAs and RNA binding proteins bring about gene silencing by recruiting the CCR4-NOT deadenylase complex to target mRNAs. Others, such as the RNA binding protein MARF1 endoribonuclease, internally cleave targeted mRNAs to engender their decay. Both the miRNA machinery and MARF1 interface with mRNA decapping factors, which in turn can enhance or inhibit their activities, respectively. Many decapping factors associate with each other through a network of multivalent interactions. These protein-protein interaction networks can in turn can lead to formation of dynamic ribonucleoprotein granules in cells called processing bodies, or `P'-bodies. How these P-bodies and their protein-protein interactions help regulate mRNA biology is not well known and is a topic of significant research. Here, I propose a research program that aims to shed light upon how the mRNA decapping machinery regulates different classes of mRNA silencing programs in the context of RNA granule integrity. We hypothesize that (i) mRNA decapping factors inhibit MARF1 endonuclease activity and (ii) modulating RNA granule integrity in turn regulates MARF1- and microRNA-mediated gene silencing programs, as well those mediated by RNA binding proteins that recruit the CCR4-NOT deadenylase complex. To test these hypotheses, we propose the following aims: 1.To elucidate the molecular underpinnings of how MARF1 interacts with the decapping machinery and how it targets select mRNAs for decay. 2.To determine how P-body granule integrity regulates MARF1-mediated mRNA decay. 3.To determine how P-body integrity modulates the mode by which microRNAs repress targeted mRNAs. The proposed research program is highly significant and builds upon key published and unpublished observations our group has made over the course of our previous NSERC Discovery grant. Ultimately, we are confident that this research proposal will generate novel insights into the modes and mechanisms by which interactions between mRNA decapping and decay factors help regulate PTC silencing programs in mammalian cells.