Regulation of microRNA Function and Cell Proliferation of Mouse Embryonic Stem Ce

描述(由申请人提供)：microRNAs(MiRNAs)是一类丰富的小RNA，可抑制靶信使RNAs(MRNAs)的表达。MiRNAs调节多种生物过程，对哺乳动物的发育至关重要，并与许多疾病有关。尽管miRNAs调控哺乳动物基因组的50%，但miRNA沉默途径的确切机制和组成仍然知之甚少。为了发挥它们的调节功能，miRNAs被掺入一种名为miRNA诱导沉默复合体(MiRISC)的核糖核蛋白复合体中，该复合体被引导到mRNA靶点。MiRNA诱导的沉默的影响是多样的；虽然一些靶标经历了降解，但另一些靶标在翻译上受到抑制。越来越明显的是，更多的蛋白质辅助因子可以与miRISC相互作用，并调节miRISC活动的性质和有效性。在这些蛋白质辅助因子中，最近发现控制各种生物的发育和干细胞命运的TRIM-NHL蛋白家族成员可以调节miRNA的功能。我们已经确定Trim71是TRIM-NHL家族中一个发育调节的成员，是一个新的miRISC相互作用蛋白，可以增强miRNAs的功能，对小鼠胚胎干细胞的快速增殖至关重要。我们的目标是从机制上深入了解Trim71在小鼠ES细胞中miRNA介导的基因抑制和细胞增殖中的作用。我们的初步数据表明，Trim71是ES细胞中一个大的多亚单位核糖核蛋白复合体的组成部分。我们将使用各种方法来鉴定含有Trim71的复合体的蛋白质和RNA成分。此外，我们将对Trim71进行详细的生化分析，从功能上确定与miRISC相互作用、增强miRNA功能和控制ES细胞增殖所需的重要蛋白质结构域。实现这一建议的目标将提供对干细胞如何在分子水平上进行调控的更好理解，并可能有助于新疗法的开发。 与公共卫生相关：干细胞为开发抗击疾病的新方法带来了巨大的希望。然而，干细胞自我更新和分化的分子基础目前还知之甚少。这项拟议的工作将为胚胎干细胞中基因表达的转录后调控提供新的见解，并可能导致操纵microRNAs的新疗法。这些研究与癌症、糖尿病、发育障碍和许多退行性疾病的治疗有关。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are an abundant class of small RNAs that repress the expression of target messenger RNAs (mRNAs). MiRNAs regulate a variety of biological processes, are essential for mammalian development and have been implicated in numerous diseases. Despite the fact that miRNAs regulate up to 50% of the mammalian genome, the exact mechanism and components of the miRNA-silencing pathway are still poorly understood. To exert their regulatory function, miRNAs are incorporated into a ribonucleoprotein complex termed miRNA- induced silencing complex (miRISC) that is guided to mRNA targets. The effects of miRNA- induced silencing are diverse; while some targets undergo degradation others are translationally repressed. It has become increasingly evident that additional protein cofactors can interact with miRISC and modulate the nature and efficacy of miRISC activity. Among these protein cofactors, members of the TRIM-NHL protein family that control development and stem cell fate in various organisms were recently found to modulate miRNA function. We have identified Trim71, a developmentally regulated member of the TRIM-NHL family, as a novel miRISC- interacting protein that enhances the function of miRNAs and is critical for rapid proliferation of mouse embryonic stem (ES) cells. Our goal is to gain mechanistic insight into the role of Trim71 in miRNA-mediated gene repression and cell proliferation in mouse ES cells. Our preliminary data indicates that Trim71 is a component of a large multi-subunit ribonucleoprotein complex in ES cells. We will employ a variety of approaches to identify the protein and RNA components of Trim71-containing complexes. In addition, we will perform a detailed biochemical analysis of Trim71 to functionally define important protein domains required for interaction with miRISC, enhancement of miRNA function and control of ES cell proliferation. Accomplishing the goals of this proposal will provide a better understanding of how stem cells are regulated at the molecular level and could aid in the development of novel therapeutics. PUBLIC HEALTH RELEVANCE: Stem cells hold great promise for the development of new approaches to combat disease. However, the molecular basis for stem cell self-renewal and differentiation are currently poorly understood. The proposed work will provide novel insight into post-transcriptional regulation of gene expression in embryonic stem cells, and may lead to new therapies to manipulate microRNAs. These studies are relevant to the treatment of cancer, diabetes, developmental disorders and numerous degenerative diseases.