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Discovery of Stress Granule Regulators and Identifying Novel Roles of Stress Granule Protein OTUD4 to Modulate ALS associated Phenotypes

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Stress granules (SGs) are highly dynamic cytoplasmic assemblies of ribonucleoprotein complexes. By stalling mRNA translation and sequestering aggregation-prone proteins, these transient membraneless organelles are thought to be a protective response during cellular stress. Defects in SG assembly and clearance are linked to neurodegenerative disease: stable SG-like inclusions in brain are hallmarks of sporadic amyotrophic lateral sclerosis (ALS) and related disorders, and genetic mutations in SG proteins cause familial forms of these diseases. Recent efforts using biochemical fractionation and protein proximity-labeling techniques have identified over 400 components in SGs. However, it has yet to be determined which proteins are critical for the formation, stabilization, or disassembly of these macromolecules, and whether they may be exploited to attenuate the progression of associated diseases. For my dissertation, I developed a screening platform combining high-content imaging on microraft arrays and pooled CRISPR knockout libraries to systematically evaluate over 1,000 RNA-binding proteins (RBPs) for roles in regulating SG abundance. This easy-to-use workflow is inexpensive and can be adapted to study gene function in the regulation of a wide range of subcellular phenotypes. With this platform, we identified and validated previously uncharacterized RBPs that regulate sodium arsenite-induced SGs when depleted. Next, we used previously published research on the composition of neuronal SGs to examine the effect of depleting specific SG components on neurodegenerative symptoms in animal models of ALS. We discovered new functions of SG proteins (DAZAP1, FAM98A, OTUD4, and SND1) in reducing pathogenic TDP-43 aggregates in human cells and improving symptoms in Drosophila models of ALS. Notably, SG protein OTUD4 was found to have a positive impact on reversing key characteristics of ALS across all models studied. Our research using both human and Drosophila models provide a comprehensive understanding of the connection between SGs and functional decline seen in ALS patients.

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This item is under embargo until April 24, 2025.