Modeling THO complex neurodevelopmental disorder mutations in gene expression regulation using budding yeast

使用芽殖酵母对基因表达调控中的 THO 复杂神经发育障碍突变进行建模

• 批准号: 10750180
• 负责人: Theresa Faith Wechsler
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750180
• 关键词: Address; Affect; Age; Biological Models; Biology; Cell physiology; Cells; Clinical; Complex; Data; Development; Disease; Disease model; Event; Family; Foundations; Gene Deletion; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome Stability; Goals; Health; Healthcare Systems; Human; Imaging Techniques; Impairment; Individual; Knowledge; Lead; Link; Messenger RNA; Metabolism; Methodology; Methods; Modeling; Molecular; Mutation; National Institute of Neurological Disorders and Stroke; Nerve Degeneration; Neurodevelopmental Disorder; Neurons; Nuclear; Nuclear Export; Outcome; Pathology; Pathway interactions; Patients; Phase; Point Mutation; Population; Protein Dynamics; RNA metabolism; RNA-Binding Proteins; Regulation; Role; Saccharomyces cerevisiae; Saccharomycetales; Scientific Advances and Accomplishments; Shapes; System; Techniques; Time; Training; Transcript; Transcription Elongation; Work; Yeast Model System; Yeasts; career; effective therapy; imaging approach; innovation; live cell imaging; messenger ribonucleoprotein; mutant; nervous system disorder; novel; programs; protein complex; protein function; recruit; reduce symptoms; skills; spatiotemporal; tool; transcriptome; transcriptome sequencing

Project Summary Fidelity of the gene expression program, involving precise temporal and spatial regulation, is critical to cellular function and organismal development. As such, perturbations or disruptions in the mechanisms governing gene expression are often implicated in disease. Neurons appear particularly sensitive to disturbances in gene expression with disruptions in RNA metabolism common in neurological diseases. For example, mutations in various RNA binding proteins (RBPs) involved in nuclear mRNA processing and export are linked to neurodevelopmental disorders (NDDs). One such complex with mutations in multiple subunits tied to NDDs is the THO complex. THO is a highly conserved complex with diverse roles in transcription and mRNA processing, with models suggesting it serves as an interaction hub for coordinating nuclear processing events. Despite these connections to NDDs and centrality to mRNA metabolism, there is still much not known regarding the dynamics of the THO complex and how disruptions in individual components impact gene expression. This knowledge gap necessitates work characterizing the role of the THO complex in gene expression. The central hypothesis of the work proposed here is that THO is critical to coordinating nuclear mRNA metabolism and disruptions in complex function that include NDD-linked mutations lead to altered dynamics of mRNA processing and gene expression outcomes. To address this hypothesis, this project will investigate the dynamics of the THO complex and model impacts of THO mutants in S. cerevisiae. Given the structural and functional conservation of the THO complex, budding yeast is a powerful model system to address this hypothesis utilizing innovative methods that would be extremely time intensive, expensive, and technically challenging in other systems. Specifically, this project employs a novel live cell imaging approach which can track recruitment of RBPs to a transcriptionally active locus over time. Utilizing this technique in combination with other approaches, THO function will be characterized in Aim 1 by temporally characterizing co-transcriptional recruitment of RBPs to a transcriptionally active locus. The outcome of these efforts will be a quantitative framework for recruitment of the THO complex relative to other RBPs. In Aim 2, the impact of complete gene deletions and disease-linked THO mutants on co- transcriptional RBP recruitment dynamics and global gene expression will be assessed. This will clarify the role of THO subunits in mRNP assembly, identify how disruptions in THO subunits shape global gene expression, and functionally characterize a subset of NDD associated point mutants. Completion of these aims will provide models that can be used to generate informed hypotheses for mechanisms by which THO complex mutations contribute to neurological disease. This information is expected to provide a critical foundation for advancing our understanding of clinically identified mutations in RBPs associated with NDDs.

项目摘要 基因表达程序的保真度涉及精确的时间和空间调节，对细胞至关重要 功能和生物发展。因此，控制基因的机制的扰动或破坏 表达通常与疾病有关。神经元似乎对基因障碍特别敏感 在神经系统疾病中常见的RNA代谢中有破坏的表达。例如，突变 参与核mRNA加工和导出的各种RNA结合蛋白（RBP）与 神经发育障碍（NDDS）。一个与NDD相关的多个亚基中突变的复合物之一是 综合体。这是一种高度保守的复合物，在转录和mRNA加工中具有不同的作用， 模型表明它是协调核加工事件的交互中心。尽管如此 与NDD的连接以及与mRNA代谢的中心性，关于动态仍然不知道 复合物以及单个成分的破坏如何影响基因表达。这个知识差距 需要表征THO复合物在基因表达中的作用的工作。中心假设 这里提出的工作是，这对于协调核mRNA代谢和复杂的破坏至关重要 包括NDD连接突变的功能 导致mRNA加工和基因表达的动力学改变 结果。为了解决这一假设，该项目将研究复合物和模型的动态 突变体在酿酒酵母中的影响。鉴于复合物的结构和功能保护， 萌芽酵母是一种强大的模型系统，可利用创新方法来解决这一假设 在其他系统中，非常耗时，昂贵且技术挑战性。具体来说，这个项目 采用一种新型的活细胞成像方法，可以跟踪RBP的募集到转录活性 随着时间的流逝。将此技术与其他方法结合在一起，将表征功能 在AIM 1中，通过将RBP的共转录募集到转录活性基因座中，通过时间表1。 这些努力的结果将是用于招募综合体相对于的定量框架 其他RBP。在AIM 2中，完全基因缺失和疾病相关的突变体对共同的影响 将评估转录RBP募集动力和全球基因表达。这将阐明角色 MRNP组装中的Tho亚基，确定亚基中的破坏如何塑造全局基因表达， 并在功能上表征了NDD相关点突变体的子集。这些目标的完成将提供 可用于生成有关复杂突变的机制的知情假设的模型 有助于神经疾病。期望此信息为推进我们的关键基础 了解与NDD相关的RBP中临床鉴定的突变。