Tuesday, May 20, 2025
5/20/2025
Structure, Function, and Dynamics of Non-Canonical Stress Granules - SUMMARY/ABSTRACT Stress granules (SGs) are dynamic, membrane-less cytoplasmic condensates of proteins and mRNAs that form in response to stressful stimuli. SGs have been linked to a broad variety of cellular processes and disease states and thus are of broad biological importance. However, much remains unknown about the structure and function of SGs. SG composition is specific to stress type, cell type, and disease state. So called “canonical” (Type I) SGs form downstream of integrated stress response activation, and are thought to be anti- apoptotic, such as those caused by acute arsenite exposure and heat shock. Other stress conditions, such as ultraviolet (UV) radiation, cause the formation of “non-canonical” SGs (Type II) with altered composition, unique kinetics, and unknown function. Many studies have thoroughly documented the composition and dynamics of canonical SGs caused by arsenite and heat. However, similar studies of non-canonical subtypes, like UV SGs, have not been performed. Our preliminary data concur with reports that UV SGs are essentially lacking in poly(A)+ RNA. We further observe for the first time that UV SGs lack the apoptotic scaffolding protein RACK1. Our results suggest that UV SGs, unlike canonical SGs, may be pro-apoptotic. Several studies have suggested that RNA-RNA and RNA-protein interactions contribute importantly to the liquid-liquid phase separation (LLPS) required for SG formation. However, if it is true that UV SGs do not contain a significant amount of RNA, this creates a paradox surrounding the role of RNA in SG formation. Our overarching hypothesis is that UV SGs represent a sort of “minimal SG” in that they appear to contain fewer proteins and RNA components than canonical SGs. We reason that UV SGs can be used as a model to understand the protein-protein interactions that drive SG formation. The goal of the proposed research is to define the composition, understand the mechanism of formation, and determine the function with respect to apoptosis, of UV SGs. To achieve these goals, we propose the following specific aims: Aim 1: Characterize the components of non-canonical UV SGs. We will test the working hypothesis that UV SGs represent a “minimal SG” and contain fewer proteins and RNAs than canonical arsenite-induced SGs, utilizing a SG purification strategy in tandem with live and fixed cell imaging. Aim 2: Elucidate the relationship between non-canonical UV SGs and apoptosis. Our working hypothesis is that phase separated UV SGs do not sequester apoptotic factors and therefore confer no benefit to the cell with respect to apoptosis. The studies proposed here are significant because they address several key unanswered questions in SG biology, including the composition of non- canonical SGs and the dynamic relationship between SG subtypes and apoptosis. Our approach is innovative in that we are utilizing SG subtypes as a mechanism to elucidate the role of RNA phase separation and protein-protein interactions within SGs in a way that has not, to our knowledge, been proposed.
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