Clearance of cytoplasmic RNA protein and mRNA-protein (mRNP) granules maintains homeostasis andprevents the accumulation of toxic species. Stress granules (SGs) and P-bodies (PBs) are mRNP granulesenriched in mRNAs RNA binding proteins and signaling proteins that often aid cell survival during stress. Thismay reflect regulation of the transcriptome and signaling pathways. Aberrant SG clearance is implicated in manycancers 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 likelycleared by various disassembly and degradative means with roles for chaperones the proteasome and aselective 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 (distinctfrom autophagy) which when induced suppresses TDP-43 toxicity. Understanding of the mechanisms andconsequences for SG PB and TDP-43 clearance remains at an early stage. It is also known that large amountsof RNA decay occur in vacuoles and lysosomes though the RNA molecules targeted trafficking mechanismsused and impacts of such decay on gene expression are unknown. Key gaps in understanding includedetermining how different clearance pathways function co-operate and affect the degradation or disassembly ofmRNP granules cytoplasmic RNA and TDP-43. The impact of such clearance pathways on cell function anddisease also requires elucidation. The aims of this grant are: 1.) define the usage importance and co-operativityof reported SG and PB clearance mechanisms under disease-relevant stress and identify the mechanism ofgranulophagy; 2.) determine the extent specificity and trafficking mechanism(s) underlying vacuolar/lysosomalRNA decay; 3.) mechanistically assess TDP-43 endolysosomal degradation and evaluate consequences toneuronal and TDP-43-related RNA phenotypes. Using genetic biochemical and cell biology assays agranulophagy model based on a prior unbiased yeast screen will be tested. These efforts will be aided by a novelSG purification method which will identify SG-localized granulophagy effectors. RNA-sequencing and vacuoleisolation will be combined to quantify the vacuolar RNA degradome while genetics and single-molecule imagingwill identify RNA vacuolar decay trafficking mechanism(s). Finally supported by an unbiased yeast screenidentifying regulators of TDP-43 abundance a model of TDP-43 degradation involving endosomal membraneinvagination will be tested. Yeast human and neuronal cell models will be used. This proposal is innovative inthat it will generate basic understanding of how novel vacuolar/lysosomal trafficking mechanisms affect RNA andprotein homeostasis. The value of this work is that the knowledge obtained will offer paradigms for clearance ofsimilar cellular substrates and globally reveal targets of an unappreciated RNA decay pathway. Finallyunderstanding clearance of SGs and cytoplasmic TDP-43 may identify therapeutic targets in ALS and cancer.