Systematic Identification of Dynamic MicroRNA Targets in Development and Stress

描述（由申请人提供）：microrna （miRNA）调节许多生物过程，miRNA表达或功能的改变与人类疾病相关，包括各种癌症、心脏病和糖尿病。然而，特定mirna调节复杂细胞和生物体功能的分子机制尚不清楚，这主要是因为通过实验鉴定的mirna的直接靶标很少。通过计算方法对miRNA靶标的预测变化很大，而且还不可靠。在这个提议中，我描述了一种新的生化方法来系统和直接鉴定miRNA靶点。通过生化分离miRNA介导的基因沉默所需的RNP复合体，该复合体既包含miRNA，也包含其主动抑制的miRNA靶点，我将同时鉴定真正的miRNA靶点，并监测miRNA的动态表达。该方法将在秀丽隐杆线虫的体内应用，以确定特定组织、发育过程和生物体应激反应中的miRNA靶点，以解决miRNA介导的基因调控是高度动态和严格调控的假设。本研究旨在实现两个主要目标：1)鉴定介导关键发育决策和机体应激反应的miRNA及其靶标；2)完善我们对指导miRNA靶基因选择的机制的理解。这些研究中产生的数据将深入了解miRNA调节增殖、分化和应激反应等过程的机制，以及miRNA功能障碍如何导致疾病状态。该提案旨在确定转录后基因调控的机制，介导重要的细胞功能，如增殖和分化。这些基因调控机制的破坏与癌症、心脏病和糖尿病等人类疾病有关。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) regulate many biological processes, and altered miRNA expression or function is associated with human diseases including a variety of cancers, heart disease, and diabetes. However, the molecular mechanisms by which specific miRNAs regulate complex cellular and organismal functions is poorly understood, largely because very few direct targets of miRNAs have been experimentally identified. Predictions of miRNA targets by computational methods are widely variable and not yet reliable. In this proposal, I describe a novel biochemical method for the systematic and direct identification miRNA targets. Through biochemical isolation of an RNP complex, shown to be required for miRNA-mediated gene silencing, that contains both miRNAs and their actively repressed miRNA targets, I will simultaneously identify bona fide miRNA targets and monitor the dynamic expression of miRNAs. This methodology will be employed in vivo in Caenorhabditis elegans to identify miRNA targets in specific tissues, during development, and in response to organismal stresses to address the hypothesis that miRNA-mediated gene regulation is highly dynamic and tightly regulated. The studies in this proposal are intended to achieve two major goals: 1) to identify miRNAs and their targets that mediate key developmental decisions and organismal stress responses, and 2) to refine our understanding of the mechanisms that direct miRNA target gene selection. The data generated in these studies will provide insight into the mechanisms by which miRNAs regulate processes such as proliferation, differentiation, and stress responses, and how miRNA dysfunction can lead to disease states. This proposal seeks to identify mechanisms of post-transcriptional gene regulation that mediate important cellular functions such as proliferation and differentiation. Disruption of these mechanisms of gene regulation is associated with human diseases such as cancer, heart disease, and diabetes.