Molecular mechanisms of microRNA mediated regulation

描述(由申请人提供)： 摘要microRNAs是一种转录后调节基因表达的~22nt的小RNA。它们有可能调节很大一部分人类基因，并与生物学的多个方面有关，从胚胎发育到细胞生理学和疾病。尽管miRNAs在基因调控中非常重要，但miRNAs调控基因表达的机制仍然存在争议。已经提出了三种miRNA介导的抑制模型：翻译起始抑制、翻译延伸抑制和mRNA去烯化。这项建议旨在利用体内系统对不同事件的相对时间(目标1，2)和干扰mRNA去烯化对miRNA介导的基因调控的分子效应(目标2)进行系统的分析。在这项研究中，我们使用斑马鱼胚胎作为体内系统，通过比较野生型胚胎和胚胎突变体在miRNA加工途径中的作用，来了解内源miRNAs在其靶标中的分子效应。利用该系统中的高通量测序，我们建议：i)通过使用核糖体足迹法测量mRNA中的核糖体密度来进行基因组范围的翻译分析(目标1)；ii)分析在存在和不存在内源miRNAs的情况下死烯基化的动态(目标2)；以及iii)分析阻断死烯基化机制对miRNA介导的翻译抑制和mRNA衰退的影响(目标2)。这些实验将使我们能够确定事件的顺序，以及在miRNA介导的基因调控中，mRNA去烯化和翻译抑制的相对贡献。MicroRNAs与多种发育缺陷、神经系统疾病以及包括肿瘤形成和转移在内的人类疾病有关。这一提议的结果将提供对miRNAs介导的基因调控所需的分子机制的基本见解，并有可能揭示miRNA途径中的重要成分，这些成分可能被用作治疗人类疾病和癌症的靶点。 公共卫生相关性： MicroRNAs是基因组中最微小的基因，与人类发育、癌症和其他人类疾病有关。这项提议旨在了解这些microRNAs调节细胞中其他基因的机制，这可能有助于我们开发特定的方法来调节它们在人类疾病期间的活动。

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.