Clearance of cytoplasmic RNA, protein and mRNA-protein (mRNP) granules maintains homeostasis and prevents the accumulation of toxic species. Stress granules (SGs) and P-bodies (PBs) are mRNP granules enriched in mRNAs, RNA binding proteins and signaling proteins, that often aid cell survival during stress. This may reflect regulation of the transcriptome and signaling pathways. Aberrant SG clearance is implicated in many cancers, viral infections, and Amyotrophic Lateral Sclerosis (ALS), where SGs may promote cytoplasmic mis- localization and aggregation of TAR DNA-binding protein 43 (TDP-43); this is toxic to neurons. SGs are likely cleared by various disassembly and degradative means, with roles for chaperones, the proteasome, and a selective autophagic pathway termed granulophagy. In contrast, PB clearance has barely been studied. Recently, cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomal trafficking pathway (distinct from autophagy), which, when induced, suppresses TDP-43 toxicity. Understanding of the mechanisms and consequences for SG, PB and TDP-43 clearance remains at an early stage. It is also known that large amounts of RNA decay occur in vacuoles and lysosomes, though the RNA molecules targeted, trafficking mechanisms used and impacts of such decay on gene expression are unknown. Key gaps in understanding include determining how different clearance pathways function, co-operate and affect the degradation or disassembly of mRNP granules, cytoplasmic RNA and TDP-43. The impact of such clearance pathways on cell function and disease also requires elucidation. The aims of this grant are: 1.) define the usage, importance and co-operativity of reported SG and PB clearance mechanisms under disease-relevant stress, and identify the mechanism of granulophagy; 2.) determine the extent, specificity and trafficking mechanism(s) underlying vacuolar/lysosomal RNA decay; 3.) mechanistically assess TDP-43 endolysosomal degradation and evaluate consequences to neuronal and TDP-43-related RNA phenotypes. Using genetic, biochemical and cell biology assays, a granulophagy model based on a prior unbiased yeast screen will be tested. These efforts will be aided by a novel SG purification method, which will identify SG-localized granulophagy effectors. RNA-sequencing and vacuole isolation will be combined to quantify the vacuolar RNA degradome, while genetics and single-molecule imaging will identify RNA vacuolar decay trafficking mechanism(s). Finally, supported by an unbiased yeast screen identifying regulators of TDP-43 abundance, a model of TDP-43 degradation involving endosomal membrane invagination will be tested. Yeast, human, and neuronal cell models will be used. This proposal is innovative in that it will generate basic understanding of how novel vacuolar/lysosomal trafficking mechanisms affect RNA and protein homeostasis. The value of this work is that the knowledge obtained will offer paradigms for clearance of similar cellul...