Molecular Mechanisms of microRNA and miRISC turnover

 描述(申请人提供)：microRNAs是一类内源性小RNA，与ArgAerte蛋白结合形成microRNA诱导沉默复合体(MiRISC)。MiRISC中的microRNAs负调控互补蛋白编码基因，以控制正常发育的许多方面。对microRNAs和miRISC的生物发生进行了广泛的研究，但对它们的周转了解相对较少。MicroRNAs 3‘端的非模板核苷酸加成与不稳定相关，但它们的功能作用尚不清楚。本工作的目的是阐明microRNA和miRISC周转的机制，并研究3‘核苷酸加成在这些过程中的作用。首先，我将在microRNA生物发生的急性失活的背景下，研究候选基因在3‘核苷酸加成和microRNA衰退中的功能。其次，我将建立一个系统来测量内源表达的ArgAerte的营业额，并使用这个系统来评估microRNA和miRISC营业额的耦合或独立程度。第三，我将进行正向基因筛查，以确定影响比率的因素 微核糖核酸的周转率。总之，这些研究将加深我们对microRNAs和miRISC丰度是如何调控的理解，并开辟多个新的研究方向。我是唯一有资格进行这项研究的人，在我的研究生和博士后培训期间，我学习了microRNA调节和生物学的不同方面。在维克多·安布罗斯的实验室里，我使用线虫模型系统发现了一种新的转录后调节microRNA活性的机制。在提议的工作中，我将应用类似的 我不仅学习了分析microRNA和miRISC营业额的技术，同时还利用了马萨诸塞大学医学院对RNA生物学的独特关注，通过与我的顾问委员会的合作，扩展了我的技能集。我的短期职业目标是通过正式的课程和实践培训学习生物化学和细胞生物学的新技术，并在过渡到独立职位的同时进行拟议的研究。我的长期目标是在这些项目的基础上建立一个多方面的研究项目，并在一家学术机构担任终身教职。

 DESCRIPTION (provided by applicant): MicroRNAs are a class of endogenous small RNAs that are bound by Argonaute proteins to form the microRNA-Induced Silencing Complex (miRISC). MicroRNAs in miRISC negatively regulate complementary protein-coding genes to control many aspects of normal development. The biogenesis of microRNAs and miRISC is extensively studied, but comparatively little is understood about their turnover. Untemplated nucleotide additions at the 3' end of microRNAs are correlated with destabilization, but their functional role remains unclear. The objective of this work is to elucidate the mechanisms of microRNA and miRISC turnover, and to examine the role of 3' nucleotide additions in these processes. First, I will examine the function of candidate genes in 3' nucleotide additions and microRNA decay in the context of acute inactivation of microRNA biogenesis. Second, I will establish a system to measure the turnover of endogenously-expressed Argonaute, and use this system to assess the extent to which microRNA and miRISC turnover are coupled or independent. Third, I will conduct forward genetic screens to identify factors that impact the rate of microRNA turnover. Together, these studies will deepen our understanding of how the abundance of microRNAs and miRISC is regulated, and open up multiple new directions of research. I am uniquely qualified to conduct this research, having studied different aspects of microRNA regulation and biology throughout my graduate and postdoctoral training. In Victor Ambros's laboratory, I have used the C. elegans model system to discover a novel mechanism of post-transcriptional regulation of microRNA activity. In the proposed work, I will apply similar techniques to dissecting microRNA and miRISC turnover, while also taking advantage of the unique focus on RNA biology at the University of Massachusetts Medical School to expand my skill set through collaboration with my advisory committee. My short term career goals are to learn new techniques in biochemistry and cell biology through formal coursework and practical training, and to carry out the proposed research while transitioning to an independent position. My long term goal is to build a multi-faceted research program on the foundation of these projects in a tenure-track position at an academic institution.