mRNA regulatory functions of the Drosophila TRIM-NHL protein, Brat

项目摘要/摘要： TRIM-NHL蛋白(以其三方基序和创始成员NCL-1、HT2A、LIN-41命名)是 对动物发育和控制干细胞命运至关重要。它们的功能障碍会导致癌症、不孕不育和 包括先天性脑积水在内的神经系统疾病。新近发现多发性TRIM-NHL 与mRNAs结合的蛋白质提供了对其分子功能的重要洞察。这样做的长期目标是 研究旨在发现TRIM-NHL蛋白如何调节基因表达以控制关键的发育 流程。这项建议的重点是果蝇黑腹果蝇Trim-NHL蛋白，脑瘤(Brat)， 它与特定的mRNAs结合，在神经发生、卵子发生和胚胎发生中发挥作用。在干细胞中， Brat抑制茎和促进分化，而功能丧失导致细胞过度增殖 大脑和生殖系。我们坚持中心假说，即Brat通过 导致mRNA降解和抑制翻译以控制干细胞的命运。我们的研究计划将决定 BRAT在神经发生和卵子发生过程中对翻译、信使核糖核酸衰变和干细胞命运的影响。一位少校 我们工作的优势在于它整合了Brat的分子调控机制和它的生物学机制 在生物体层面上发挥作用。首先，使用专门测量Brat活动的定量分析，我们将 剖析其抑制域，并确定培养细胞中所需的途径和辅阻遏子。第二, 我们将确定由Brat调控的基因网络。我们将绘制整个Brat绑定站点的地图 转录组学和测量BratʹS对表达的mRNA丰度和翻译状态的影响。通过 整合产生的数据，我们的数据将提供Brat-RNA之间关系的全面视图 功能结合位点的位置，以及对信使核糖核酸稳定性和翻译效率的影响。第三, 我们使用精确的基因组编辑来询问幼虫的RNA结合和抑制结构域的作用 果蝇神经发生和卵子发生过程中的基因。我们还创造了组织特异性报告基因分析 这专门测量了BratʹS在干细胞中的抑制活性。这项研究的结果将提供一个 从机理上理解Brat介导的基因调控，并提供其对基因影响的全球视角 表情。我们的结果将确定Brat的mRNA调节活动在干细胞控制中的作用 增殖分化轴。Brat是TRIM-NHL家族的原型，我们的发现将 也广泛地提高了对TRIM-NHL蛋白在发育和疾病中的功能的理解。

Project Summary/Abstract: TRIM-NHL proteins (named after their TRIpartite Motif and founding members NCL-1, HT2A, LIN-41) are essential for animal development and control of stem cell fate. Their dysfunction causes cancer, infertility, and neurological disorders including congenital hydrocephalus. The recent discovery that multiple TRIM-NHL proteins bind to mRNAs provided a crucial insight into their molecular functions. The long term goal of this research is to discover how TRIM-NHL proteins regulate gene expression to control crucial developmental processes. This proposal focuses on the Drosophila melanogaster TRIM-NHL protein, Brain Tumor (Brat), which binds to specific mRNAs and functions in neurogenesis, oogenesis, and embryogenesis. In stem cells, Brat suppresses stemness and promotes differentiation whereas loss-of-function leads to over-proliferation in the brain and germline. We pursue the central hypothesis that Brat negatively regulates gene expression by causing mRNA degradation and inhibiting translation to control stem cell fate. Our research plan will determine the effect of Brat on translation, mRNA decay, and stem cell fate during neurogenesis and oogenesis. A major strength of our work is that it integrates both molecular regulatory mechanisms of Brat and its biological functions at the organismal level. First, using quantitative assays that specifically measure Brat activity, we will dissect its repressive domains and identify the required pathways and corepressors in cultured cells. Second, we will identify the network of genes that are regulated by Brat. We will map Brat-binding sites across the transcriptome and measure Bratʹs effect on the abundance and translation status of expressed mRNAs. By integrating the resulting data, our data will provide a comprehensive view of the relationship between Brat-RNA occupancy, location of functional binding sites, and impact on mRNA stability and translation efficiency. Third, we use precision genome editing to interrogate the roles of RNA-binding and repression domains of the brat gene during neurogenesis and oogenesis in Drosophila. We also created tissue specific reporter gene assays that specifically measure Bratʹs repressive activity in stem cells. The results of this research will provide a mechanistic understanding of Brat mediated gene regulation and provide a global view of its impact on gene expression. Our results will establish the role of Brat’s mRNA regulatory activities in the control of the stem cell proliferation-differentiation axis. Brat serves as an archetype for the TRIM-NHL family, and our discoveries will also broadly enhance the understanding of TRIM-NHL protein functions in development and disease.