PROJECT SUMMARY Identification of two RNA-binding proteins (RBPs), FUS and TDP-43, as causative factors of Amyotrophic Lateral Sclerosis (ALS) resulted in a paradigm shift centered on RNA dysfunction as a disease-driving mechanism. We previously established a yeast model of FUS cytotoxicity. Using this model, we carried out genome-wide overexpression screen and identified five yeast genes that rescue FUS toxicity. Three out of five suppressor genes have human homologs and all three encode RBPs. We found that hUPF1, the human homolog of one of the three RBPs, rescues the toxicity of FUS in mouse primary neurons and a rat model of ALS. These findings support the value of our study of FUS toxicity in yeast and uncovered a novel pathway, currently under development as new therapeutics for ALS. Motivated by this success, we constructed a new genome-scale library, containing 13,570 full-length sequence verified human gene clones individually cloned in an inducible yeast-expression vector. Using this library and a newly developed efficient screening method, we identified 37 human genes, when overexpressed, robustly rescuing FUS induced toxicity in yeast. Genes encoding RBPs are highly enriched among suppressors (12 out of 37). Strikingly, all 12 RBP suppressors have known connections to stress granules (SG). The objective of this application is to define mechanisms of FUS toxicity by studying how the 12 RBPs work to rescue cellular defects induced by FUS. We hypothesize that toxicity of FUS involves direct impairment of SG function, and human RBP suppressors rescue FUS toxicity by alleviating detrimental effect of FUS on SG. Two specific Aims are proposed: i) examine the effect of suppressor RBPs on FUS aggregation and localization;; and ii) examine the effects of suppressor RBPs on SG. Our hypothesis is consistent with findings that FUS protein aggregates mis-localize to SG in yeast and in mammalian cells. Based on our preliminary data on TAF15, one of the human RBP suppressors, we reason that the suppressor mechanisms likely involve interactions between FUS and the suppressors as well as alterations in SG structure and function. Regulation and mis-regulation of SG is a pathway that is of immense interest to ALS research field. Completion of this project will provide deeper understanding of FUS mediated cytotoxicity, its connection to RNA metabolism, particularly with respect to aberrant SG function. Projects proposed here will not only expose more undergraduate students to meritorious research in the PI’s laboratory but also help promote discussion on scientific discoveries in an upper lever undergraduate course, regularly offered by the PI. Both serve to strengthen the research environment at the PI’s home institution.