MECHANISMS FOR MODULATION OF MIRNA-MEDIATED GENE SILENCING

 描述(申请人提供)：miRNAs是基因表达的关键转录后调节因子之一，在发育、细胞增殖、细胞分化、代谢和凋亡等广泛的生物过程中发挥作用。我们先前已经推导出在miRNA诱导的翻译抑制过程中几个细胞事件的机制和相对时间。在这里，我们将重点阐明调节这一调控途径的机制。在报告和基于全球的方法中都观察到了miRNA诱导的基因调控水平的差异，这让我们问为什么会有这些差异，以及这些差异是如何存在的。利用我们的体内可诱导和可控制的报告系统，以及体外技术，我们将研究mRNA靶的内在稳定性对miRNA介导的基因沉默过程中Mirics组分的动力学和解剖贡献的影响。我们将利用这个系统和各种修饰的和天然的报告基因来更全面地了解不同的RNA结合蛋白和miRNA途径组件之间在基因调控方面的关系。我们将使用我们新开发的分析和技术来推断这些RNA结合蛋白调控miRNA介导的基因沉默的分子机制。最后，在这些研究之后，将在生物学背景下采用全基因组方法，使我们能够调查顺式和反式因子对跨内源基因靶标的特定miRNA反应调节的重要性。除了极大地扩展我们对miRNA介导的基因沉默的了解外，拟议的工作还将为我们提供更多线索，了解调节miRNA反应的系统的变化如何影响正常和病理状态下的细胞动态平衡。

 DESCRIPTION (provided by applicant): miRNAs are one of the key post-transcriptional regulators of gene expression and play a role in a wide range of biological processes including development, cell proliferation, cell differentiation, metabolism and apoptosis. We have previously deduced the mechanism and relative timing of several cellular events during miRNA- induced translational repression. Here, we will focus on elucidating mechanisms that modulate this regulatory pathway. Variations in the level of miRNA-induced gene regulation are observed in both reporter and global- based approaches, leading us to ask why and how these differences exist. Using our inducible and controllable in vivo reporter systems, as well as in vitro techniques, we will investigate the effects of mRNA target intrinsic stability on the kinetic and dissect contributions of miRICS components in the process of miRNA-mediated gene silencing. We will employ this system with a variety of modified and natural reporter genes to get a more complete picture of the relationships between distinct RNA binding proteins and miRNA pathway components on gene regulation. We will use our newly developed assays and techniques to deduce the molecular mechanisms for modulation of miRNA-mediated gene silencing by these RNA-binding proteins. Finally, these studies will be followed by genome-wide approaches in a biological context that will allow us to investigate the importance of cis and trans factors on the modulation of specific miRNA response across endogenous gene targets. In addition to vastly expanding our knowledge about miRNA-mediated gene silencing, the proposed work will give us more clues as to how alterations in systems that modulate miRNA response may influence cellular homeostasis in regular and pathological states.