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 基因表达程序的精确性，包括精确的时间和空间调控，对细胞生长至关重要。 功能和组织发育。因此，基因调控机制的干扰或破坏 表达往往与疾病有关。神经元似乎对基因干扰特别敏感， 在神经系统疾病中常见的RNA代谢中断的表达。例如， 参与核mRNA加工和输出的各种RNA结合蛋白（RBP）与 神经发育障碍（NDD）。其中一个与NDD相关的多个亚基突变的复合体是 THO复合体THO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处理事件的互动中心。尽管有这些 与NDD的联系和mRNA代谢的中心性，关于动力学仍然有很多未知 THO复合物的破坏以及单个组分的破坏如何影响基因表达。这一知识空白 需要研究THO复合物在基因表达中的作用。的中心假设 这里提出的工作是，THO是协调核mRNA代谢和破坏复杂 包括NDD连锁突变的功能 导致mRNA加工和基因表达动力学改变 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 THO突变体在S.啤酒。考虑到THO复合物的结构和功能保守性， 芽殖酵母是一个强大的模型系统，以解决这一假设，利用创新的方法， 在其他系统中，这是非常耗时、昂贵和技术上具有挑战性的。具体来说，这个项目 采用了一种新的活细胞成像方法，该方法可以跟踪RBP向转录活性 随时间变化的轨迹。利用这种技术与其他方法相结合，THO函数将被表征 在目的1中，通过在时间上表征RBP向转录活性基因座的共转录募集。 这些努力的结果将是一个定量框架，用于招募THO复合体， 其他RBPs。在目标2中，完整基因缺失和疾病连锁THO突变体对共 将评估转录RBP募集动力学和整体基因表达。这将阐明 THO亚基在mRNP装配中的作用，确定THO亚基的破坏如何塑造全局基因表达， 并在功能上表征NDD相关点突变体的子集。这些目标的实现将为 模型，可用于生成THO复杂突变机制的知情假设 会导致神经系统疾病这些信息有望为推进我们的 了解与NDD相关的RBP中临床鉴定的突变。