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

果蝇 TRIM-NHL 蛋白 Brat 的 mRNA 调节功能

• 批准号: 10670824
• 负责人: Aaron Charles Goldstrohm
• 金额: $ 29.44万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670824
• 关键词: Adult; Affect; Animals; BRAT gene; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological; Biological Assay; Biological Process; Brain; Brain Neoplasms; Cancer Etiology; Cell Culture Techniques; Cells; Complex; Congenital Hydrocephalus; Cultured Cells; Data; Development; Developmental Process; Disease; Drosophila genus; Drosophila melanogaster; Embryonic Development; Family; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic; Goals; Infertility; Lobular Neoplasia; Location; Malignant Neoplasms; Maps; Measures; Mediating; Messenger RNA; Modeling; Molecular; Mutate; Names; Neurodevelopmental Disorder; Oogenesis; Pathogenesis; Pathway interactions; Poly(A) Tail; Post-Transcriptional Regulation; Proliferating; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Reporter; Reporter Genes; Repression; Research; Ribonucleases; Role; TRIM Gene; TRIM Motif; Tail; Tissues; Translational Repression; Translations; Work; fly; gene network; genetic corepressor; genome editing; improved; in vivo; insight; loss of function; mRNA Decay; mRNA Expression; mRNA Stability; mRNA Transcript Degradation; mRNA Translation; member; mutant; nervous system disorder; neurogenesis; polyadenosine; protein function; stem cell fate; stem cell proliferation; stem cells; stemness; transcription factor; transcriptome; translatome

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.

项目摘要/摘要： TRIM-NHL 蛋白（以其三部分基序和创始成员 NCL-1、HT2A、LIN-41 命名）是 对于动物发育和干细胞命运的控制至关重要。它们的功能障碍会导致癌症、不孕症和 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 的结合为了解其分子功能提供了重要的见解。本次活动的长远目标 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。该提案重点关注果蝇 TRIM-NHL 蛋白、脑肿瘤 (Brat)、 它与特定的 mRNA 结合，并在神经发生、卵子发生和胚胎发生中发挥作用。在干细胞中， Brat 抑制干性并促进分化，而功能丧失则导致过度增殖 大脑和种系。我们追求的中心假设是 Brat 通过以下方式负向调节基因表达： 引起 mRNA 降解并抑制翻译以控制干细胞的命运。我们的研究计划将决定 Brat 对神经发生和卵子发生过程中翻译、mRNA 衰减和干细胞命运的影响。一个专业 我们工作的优势在于它整合了 Brat 的分子调控机制及其生物 在有机体水平上发挥作用。首先，使用专门测量 Brat 活性的定量测定，我们将 剖析其抑制域并确定培养细胞中所需的途径和辅抑制因子。第二， 我们将确定由 Brat 调控的基因网络。我们将绘制 Brat 结合位点的分布图 转录组并测量 Brat's 对表达 mRNA 丰度和翻译状态的影响。经过 整合所得数据，我们的数据将提供 Brat-RNA 之间关系的全面视图 功能结合位点的占用、位置以及对 mRNA 稳定性和翻译效率的影响。第三， 我们使用精确基因组编辑来探究小子的 RNA 结合和抑制域的作用 果蝇神经发生和卵子发生过程中的基因。我们还创建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 对 Brat 介导的基因调控的机制理解并提供其对基因影响的全局视图 表达。我们的结果将确定 Brat 的 mRNA 调节活性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 还广泛增强了对 TRIM-NHL 蛋白在发育和疾病中功能的理解。