Molecular mechanisms of microRNA mediated regulation

描述（由申请人提供）： MicroRNA是一种约22个核苷酸的小分子RNA，在转录后调节基因表达。它们具有调节大部分人类基因的潜力，并且涉及从胚胎发育到细胞生理学和疾病的生物学的多个方面。尽管它们在基因调控中至关重要，但miRNAs调控基因表达的机制仍然存在争议。目前已经提出了三种miRNA介导的阻遏模型：翻译起始阻遏、翻译延伸阻遏和mRNA去腺苷化阻遏。该提案旨在使用体内系统对不同事件的相对时间（目的1，2）和破坏mRNA去腺苷化（目的2）对miRNA介导的基因调控的分子效应进行系统分析。在这个提议中，我们使用斑马鱼胚胎作为体内系统，通过比较野生型胚胎与胚胎突变体在miRNA加工途径中的差异，来了解内源性miRNA在其靶点中的分子效应。在该系统中使用高通量测序，我们提出i）通过使用核糖体足迹法测量mRNA中的核糖体密度来进行时间基因组范围的翻译分析（Aim 1），ii）分析在存在和不存在内源性miRNA的情况下去腺苷化的动力学（Aim 2），和iii）分析阻断去腺苷化机制对miRNA介导的翻译抑制和mRNA衰减的影响（目的2）。这些实验将使我们能够确定事件的顺序，以及在miRNA介导的基因调控过程中mRNA去腺苷化和翻译抑制的相对贡献。MicroRNA与多种发育缺陷、神经系统疾病和人类疾病（包括肿瘤形成和转移）有关。从这个提议中得到的结果将为miRNA介导的基因调控所需的分子机制提供基本的见解，并有可能揭示miRNA途径中的重要组分，这些组分可用作治疗人类疾病和癌症的治疗靶点。 公共卫生相关性： MicroRNA是基因组中最小的基因，与人类发育、癌症和其他人类疾病有关。该提案旨在了解这些microRNA调节细胞中其他基因的机制，这可能有助于我们开发在人类疾病期间调节其活性的特定方法。

DESCRIPTION (provided by applicant): Summary MicroRNAs are ~22nt small RNAs that regulate gene expression post transcriptionally. They have the potential to regulate a large fraction of the human genes, and have been implicated in multiple aspects of biology from embryonic development, to cellular physiology and disease. Despite their paramount importance in gene regulation, the mechanisms by which miRNAs regulate gene expression remains remain controversial. Three models for miRNA mediated repression have been proposed: repression of translation initiation, repression of translation elongation and mRNA deadenylation. This proposal aims to undertake a systematic analysis of the relative timing of the different events (aim 1, 2) and the molecular effect of disrupting mRNA deadenylation (aim 2) on miRNA mediated gene regulation using an in vivo system. In this proposal we use the zebrafish embryo as an in vivo system to understand the molecular effect of endogenous miRNAs in their targets by comparing wild type embryos with embryos mutants in the miRNA processing pathway. Using high throughput sequencing in this system we propose to i) undertake a temporal genome wide analysis of translation by measuring ribosome density in the mRNA using Ribosome foot- printing (Aim 1), ii) analyze the dynamics of deadenylation in the presence and the absence of endogenous miRNAs (Aim 2), and iii) analyze the effect of blocking the deadenylation machinery on miRNA mediated translational repression and mRNA decay (Aim 2). These experiments will allow us to determine the sequence of events, and the relative contribution of mRNA deadenylation and translational repression during miRNA-mediated gene regulation. MicroRNAs have been implicated in a wide range of developmental defects, neurological disorders, and human disease including tumor formation and metastasis. The results derived from this proposal will provide fundamental insights into the molecular machinery required for miRNAs mediated gene regulation and has the potential to reveal important components in the miRNA pathway that may be used as therapeutic targets to treat human diseases and cancer. PUBLIC HEALTH RELEVANCE: MicroRNAs constitute the tiniest genes in the genome, and have been implicated in human development, cancer and other human diseases. This proposal aims to understand the mechanism by which these microRNAs regulate other genes in the cell, what might help us develop specific ways to modulate their activity during human disease.