Characterizing the Role of RSP5 in Stress Granule Clearance

Abstract

Stress granules (SGs) are conserved cytoplasmic condensates of non-translating mRNA and associated RNA-binding proteins. SG assembly is induced by the accumulation of non-translating mRNAs during adaptation of cells to stressful environments. While SG assembly is relatively well understood, many mechanisms including chaperone function, post-translational modifications, proteasomal and autophagic (granulophagy) degradation have been reported to function in SG clearance. However, the mechanisms, utilization and integration of these clearance pathways is poorly understood. Understanding SG clearance is important as it likely impacts post-transcriptional gene expression, and possibly other SG-related functions. Studying SG clearance may also aid in furthering our understanding of diseases such as Amyotrophic Lateral Sclerosis (ALS) and Cancer, where aberrant persistence of SGs is theorized to contribute to disease pathology. In this work, we performed an unbiased genome wide screen in Saccharomyces cerevisiae (budding yeast) to identify regulators of SG clearance via granulophagy. This screen identified several genes involved in ubiquitin-related biology, including co-factors of the essential E3 ligase Rsp5, which adds K63-linked ubiquitin chains to substrates which facilitates vacuolar/lysosomal trafficking. Using conditional genetics, we find that Rsp5 strongly promotes SG clearance after heat shock (HS) and stationary phase stress (SP), but not sodium azide stress. Additionally, our preliminary data suggests that Rsp5 colocalizes with SGs during the initial phase of stress recovery. Rsp5-mediated SG clearance represents a novel stress specific clearance mechanism, which given the presence of a clear homolog in human cells (NEDD4), could ultimately be harnessed as a therapeutic target to counter SG-associated disease pathology.