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

项目摘要 基因表达程序的保真度，包括精确的时间和空间调节，对细胞至关重要 功能和生物体发育。因此，基因控制机制中的干扰或中断 表情通常与疾病有牵连。神经元似乎对基因的干扰特别敏感 在神经系统疾病中常见的RNA代谢紊乱的表达。例如，基因突变 参与核mRNA加工和输出的各种RNA结合蛋白(RBPs)与 神经发育障碍(NDDS)。与NDDS相关的多个亚基发生突变的一个这样的复合体是 Tho情结。THO是一个高度保守的复合体，在转录和mRNA处理中具有不同的作用， 模型表明，它是协调核处理事件的互动中心。尽管如此 与NDDS的联系以及对mRNA新陈代谢的中心性，关于其动力学仍有许多未知之处 以及单个成分的中断如何影响基因表达。这种知识鸿沟 这就需要研究THO复合体在基因表达中的作用。的核心假说 这里提出的工作是，THO对于协调复合体中的核mRNA代谢和破坏是至关重要的 包括NDD连锁突变的功能 导致mRNA处理和基因表达的动态改变 结果。为了解决这一假设，本项目将研究tho复合体和模型的动力学。 突变体对酿酒酵母的影响。鉴于THO复合体的结构和功能保守， 芽殖酵母是一个强大的模型系统，可以利用创新的方法来解决这一假说，这将是 在其他系统中非常耗时、昂贵，而且在技术上具有挑战性。具体地说，这个项目 采用一种新的活细胞成像方法，可以跟踪限制性商业惯例到转录活性的募集 随时间推移的轨迹。利用这一技术与其他方法相结合，将对功能进行表征 在目标1中，通过在时间上表征限制性商业惯例向转录活性位点的共转录招募。 这些努力的结果将是与以下方面有关的THO综合体征聘的量化框架 其他限制性商业惯例。在目标2中，基因完全缺失和疾病连锁突变对联合基因突变的影响。 将评估转录RBP招募动态和全球基因表达。这将澄清其角色 在mRNP组装的亚基中，确定亚基的中断如何塑造全球基因表达， 并在功能上鉴定与NDD相关的点突变的子集。完成这些目标将提供 可用于生成复杂突变机制的知情假设的模型 会导致神经系统疾病。预计这些信息将为推进我们的 了解临床确定的与NDDS相关的限制性商业惯例突变。

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.