Genetic analysis of post-transcriptional modulators of microRNAs in C. elegans

描述（由申请人提供）：microRNA在调节动物的多种生物过程中起着至关重要的作用，但microRNA本身的调节机制在很大程度上是未知的。Ambros实验室和其他人最近的研究表明，microRNA在转录后的许多水平上受到调控，包括初级到成熟microRNA加工，RISC活性和microRNA周转。本研究旨在利用C. elegans作为模型系统，特别关注microRNA周转。首先，我将开发表达两种荧光报告基因的转基因蠕虫菌株，以通过同时跟踪microRNA靶抑制和microRNA启动子的转录活性来直接监测let-7或mir-35-41的转录后调控的变化。我将使用这些菌株进行经典的遗传筛选以及候选人驱动的RNAi筛选，以确定修饰microRNA靶抑制但不修饰主要microRNA转录的基因。其次，我将描述候选基因在microRNA周转中的作用，重点是研究XRN-2。我将对XRN-2和其他候选人进行RNAi，以确定它们对microRNA稳定性的直接影响，确定它们调节哪些microRNA，并确定它们是否因此在任何先前表征的涉及microRNA的体内过程中发挥生物学作用。本文提出的研究将开始阐明调节microRNA稳定性的机制，并可能开辟该领域多个新的研究方向。总的来说，当与其他水平的基因表达控制相结合时，我们对这些过程的基本理解对于理解人类疾病状态至关重要。 公共卫生相关性：microRNA的表达在许多人类疾病状态中是失调的，包括癌症。虽然已经深入研究了microRNA的转录和生物发生，但对microRNA的周转知之甚少，这在调节microRNA水平方面可能同样重要。因此，了解microRNA的转录后调控，特别是microRNA的周转，将是至关重要的，以促进我们的理解基因表达的变化，人类疾病的基础。

DESCRIPTION (provided by applicant): MicroRNAs play crucial roles regulating diverse biological processes in animals, but the mechanisms by which microRNAs themselves are regulated are largely unknown. Recent studies from the Ambros laboratory and others suggest that microRNAs are regulated at many levels post-transcriptionally, including primary to mature microRNA processing, RISC activity, and microRNA turnover. The research proposed here aims to elucidate post-transcriptional regulation of microRNAs using C. elegans as a model system, with a particular focus on microRNA turnover. First, I will develop transgenic worm strains that express two fluorescent reporters to directly monitor changes in post- transcriptional regulation of let-7 or mir-35-41 by simultaneously tracking microRNA target repression and transcriptional activity of the microRNA promoter. I will use these strains to conduct classical genetic screens as well as candidate-driven RNAi screens to identify genes that modify microRNA target repression but not primary microRNA transcription. Second, I will characterize the roles of candidate genes in microRNA turnover, with an emphasis on studying XRN-2. I will perform RNAi against XRN-2 and other candidates, in order to determine their direct impact microRNA stability, define which microRNAs they regulate, and determine whether they thus play a biological role in any previously-characterized process involving microRNAs in vivo. The studies proposed here will begin to elucidate the mechanisms that regulate microRNA stability, and will likely open up multiple new directions of study in the field. Overall, our basic comprehension of these processes will be crucial for the understanding of human disease states, when integrated with other levels of gene expression control. PUBLIC HEALTH RELEVANCE: The expression of microRNAs is deregulated in many human disease states, including cancer. Though the transcription and biogenesis of microRNAs has been studied in depth, very little is known about microRNA turnover, which could be equally important in regulating microRNA levels. Therefore, an understanding of post-transcriptional regulation of microRNAs, and microRNA turnover specifically, will be crucial to advancing our comprehension of gene expression changes that underlie human disease.