PROJECT SUMMARY/ABSTRACT A number of RNA binding proteins with disordered low complexity domains have recently been shown to undergo liquid-liquid phase separation which underlies membraneless organelle architecture both in cells and in vitro. Several of these proteins are mutated and form inclusions in the neurodegenerative diseases amyotrophic lateral sclerosis and multisystem proteinopathy. One such protein, heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), is a major component neuronal mRNA transport granules that carry transcripts, including immediate early gene activity-regulated cytoskeleton associated protein Arc, to sites of local translation in dendrites. Using in vitro structural studies including nuclear magnetic resonance spectroscopy and a novel disease model in C. elegans, I will determine the molecular contacts and protein-protein interactions underlying granule formation, as well as the effect of post-translational modifications and disease mutations on physiological interactions, pathological aggregation, and neurodegeneration. Aim 1 will determine the effects of the disease mutation in vitro and develop a disease model of neurodegeneration by replacing the third exon of the C. elegans gene with the corresponding human sequence. I hypothesize that the disease mutants will cause aggregation in vitro and neurodegeneration in vivo. Preliminary results have shown that hnRNPA2 undergoes liquid-liquid phase separation mediated by its low complexity domain and that disease mutants aggregate within phase-separated droplets. Aim 2 will determine the effects of post-translational modifications, including asymmetric arginine dimethylation and tyrosine phosphorylation on phase separation and aggregation as well as the genetic interaction in C. elegans of hnRNPA2 with the methyltransferase, kinase, and phosphatase responsible for the post-translational modifications. I hypothesize that tyrosine phosphorylation will decrease phase separation and aggregation of the disease mutant and that loss of each enzyme will change the degree of neurodegeneration in vivo. Aim 3 will examine the interaction between hnRNPA2 and ch-TOG (CKAP5), a microtubule binding protein that was previously shown to bind hnRNPA2 in myelin basic protein mRNA transport granules. I hypothesize that a) TOG acts as a multivalent scaffolding protein that binds hnRNPA2 at tyrosine residues, b) that tyrosine phosphorylation will disrupt this interaction, and c) that loss of TOG will increase neurodegeneration. Results from this proposal will provide critical understanding of the molecular interactions underlying granule formation, how those interactions are disrupted by disease mutations, and will lead to possible drug targets to prevent protein aggregation and associated neurodegeneration.