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

项目概要/摘要： TRIM-NHL蛋白（以其三分基序和创始成员NCL-1、HT 2A、LIN-41命名）是 对动物发育和控制干细胞命运至关重要。他们的功能障碍会导致癌症，不孕不育， 神经系统疾病，包括先天性脑积水。最近发现，多个TRIM-NHL 蛋白质与mRNA的结合提供了对其分子功能的重要见解。长期目标是 研究的目的是发现TRIM-NHL蛋白如何调节基因表达，以控制关键的发育 流程.这项提案的重点是果蝇TRIM-NHL蛋白，脑肿瘤（布拉特）， 其与特异性mRNA结合并在神经发生、卵子发生和胚胎发生中起作用。在干细胞中， Brat抑制干细胞并促进分化，而功能丧失导致细胞过度增殖， 大脑和生殖细胞我们追求的核心假设是，布拉特负调控基因表达， 导致mRNA降解并抑制翻译以控制干细胞命运。我们的研究计划将决定 Brat对神经发生和卵子发生过程中的翻译、mRNA衰变和干细胞命运的影响。一个主要 我们工作的优势在于它整合了Brat的分子调控机制和其生物学特性， 在生物体水平上发挥作用。首先，使用专门测量Brat活性的定量分析，我们将 剖析其抑制结构域，并确定所需的途径和辅阻遏物在培养的细胞。第二、 我们将确定由布拉特调控的基因网络。我们将绘制出 转录组和测量Brat对表达的mRNA的丰度和翻译状态的影响。通过 整合所得数据，我们的数据将提供一个全面的看法Brat-RNA之间的关系， 占据、功能结合位点的位置以及对mRNA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brat的RNA结合和抑制结构域的作用， 基因在果蝇神经发生和卵子发生中的作用。我们还建立了组织特异性报告基因检测 专门测量布拉特在干细胞中的抑制活性。这项研究的结果将提供一个 对Brat介导的基因调控机制的理解，并提供其对基因调控的影响的全局视图。 表情我们的研究结果将确立Brat的mRNA调控活性在干细胞调控中的作用。 增殖分化轴布拉特作为一个原型的TRIM-NHL家庭，我们的发现将 还广泛增强了对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.