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

项目总结

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.