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

描述(申请人提供)：microRNAs在调节动物体内的各种生物过程中起着至关重要的作用，但microRNAs本身被调节的机制在很大程度上还不清楚。Ambros实验室和其他实验室最近的研究表明，microRNAs在转录后的许多水平上受到调控，包括从初级到成熟的microRNA加工、RISC活性和microRNA周转。本研究旨在以秀丽线虫为模型系统，重点研究microRNA的周转，阐明microRNA的转录后调控。首先，我将开发表达两个荧光报告基因的转基因蠕虫株，通过同时跟踪microRNA靶标抑制和microRNA启动子的转录活性，直接监测let-7或mir-35-41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驱动的RNAi筛选，以确定修改microRNA靶标抑制而不是初级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和其他候选基因进行RNAi，以确定它们直接影响microRNA的稳定性，定义它们调节哪些microRNAs，并确定它们是否因此在体内涉及microRNAs的任何先前表征的过程中发挥生物学作用。这里提出的研究将开始阐明调节microRNA稳定性的机制，并可能在该领域开辟多个新的研究方向。总体而言，当我们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将对于理解人类疾病状态至关重要。 与公共卫生相关：在包括癌症在内的许多人类疾病状态下，microRNAs的表达都被解除了管制。虽然对microRNA的转录和生物发生已经进行了深入的研究，但对microRNA的周转知之甚少，它在调节microRNA水平方面可能同样重要。因此，了解microRNAs的转录后调控，特别是microRNA的周转，对于促进我们对导致人类疾病的基因表达变化的理解至关重要。

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.