Investigating how mRNA decapping factors regulate gene silencing programs

转录后控制程序(PTC)调节信使(M)RNA从DNA模板转录后蛋白质的产生。许多PTC程序通过抑制信使核糖核酸的翻译和/或启动信使核糖核酸失稳来控制蛋白质的产生，这些事件统称为“沉默”。许多PTC介体，包括微(MI)RNAs和RNA结合蛋白，通过招募CCR4-Not Deadenylase复合体靶向mRNAs而导致基因沉默。其他的，如RNA结合蛋白MARF1内切核糖核酸酶，在内部切割目标mRNA以产生它们的腐烂。MiRNA机制和MARF1都与信使核糖核酸解离因子结合，进而分别增强或抑制其活性。许多解脱因子通过多价相互作用的网络相互关联。这些蛋白质-蛋白质相互作用网络可以反过来导致在细胞中形成动态的核糖核蛋白颗粒，称为加工小体或‘P’小体。这些P小体和它们的蛋白质-蛋白质相互作用如何帮助调节mRNA生物学还不是很清楚，这是一个重要的研究课题。在这里，我提出了一个研究计划，旨在阐明在RNA颗粒完整性的背景下，信使核糖核酸解译机制如何调节不同类别的信使核糖核酸沉默程序。我们假设(I)mRNA解离因子抑制MARF1内切酶活性，(Ii)调节RNA颗粒完整性反过来调节MARF1和microRNA介导的基因沉默程序，以及由RNA结合蛋白介导的那些招募CCR4-而不是死烯基酶复合体的基因沉默程序。为了验证这些假说，我们提出了以下目标：1.阐明MARF1如何与解离机制相互作用的分子基础，以及它如何靶向选择要衰退的mRNA。2.确定P小体颗粒完整性如何调节MARF1介导的mRNA衰变。3.确定P-小体完整性如何调节microRNAs抑制靶mRNAs的方式。拟议的研究计划意义重大，并建立在我们的团队在之前的NSERC发现拨款过程中所做的关键的已发表和未发表的观察结果的基础上。最终，我们相信，这项研究建议将产生新的洞察力，通过这些模式和机制，mRNA解压和衰变因子之间的相互作用有助于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.