Stress granules (SGs) are assemblies of non-translating mRNA-protein complexes (mRNPs) that are conserved throughout eukaryotes, and which normally form only transiently when bulk translation is inhibited during cellular stress. SGs sequester mRNPs and signaling factors, and play key roles in cellular stress responses by modulating gene expression and cell signaling. Previously, we determined that SGs are cleared via a novel selective autophagic pathway that we termed granulophagy. This finding was important as it demonstrated a fundamentally new way in which SGs are cleared; previously SGs were thought to disassemble only by non-destructive means. Granulophagy thus likely impacts upon gene expression at the mRNA level, and could explain the underlying pathology of several inclusion body disorders such as Amyotrophic Lateral Sclerosis, Frontotemporal Lobar Dementia and Paget's disease. These are typically characterized by constitutive SG-like aggregates and autophagy defects in affected cells. Key gaps in our understanding include how are SGs mechanistically targeted by granulophagy, how is this process regulated, and what is the physiological relevance of granulophagy. Our long-term goal is to understand the means by which cells assemble, disassemble or clear mRNP granules, how this affects mRNA regulation, and the progression of diseases characterized by aberrant mRNP granule formation. The overall objectives of this application are to identify genes affecting granulophagy and determine protein interactions between SG and autophagy proteins that are required for granulophagy activity. We also wish to determine how granulophagy is regulated, and what the consequences of granulophagy are on both mRNA stability and clearance of SG-like protein aggregates observed in inclusion body disorders. Our central hypothesis is that selective autophagy receptor proteins interact with SGs and recruit the autophagic machinery, especially under conditions that limit SG disassembly via other means. Using yeast and cell line model systems, and proven genetic, biochemical and microscopy techniques in the SG and autophagy fields, we shall test this hypothesis with the following three aims: 1.) What genes promote granulophagy, and how? 2.) What conditions induce granulophagy, and how is this regulated? 3.) How does granulophagy affect mRNA decay and clearance of SG-like disease aggregates? This proposal is innovative, as it will enhance our understanding of a novel selective autophagic pathway that targets a physiologically important substrate (SGs) with implications in disease. This contribution is important because understanding granulophagy in greater detail may suggest new paradigms of selective autophagic function, and illuminate new modes of mRNA and cell signaling regulation relevant to both general cell biology, and to diseases characterized or caused by aberrant SG accumulation.
Aberrant persistence of SGs appears causative in a variety of degenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration (FTLD) and Pagets disease. Several pieces of evidence suggest a failure to clear SGs, and similar aggregates via autophagy, is the crucial defect underpinning pathogenesis in these diseases. Therefore a greater understanding of targeting, regulation and the functional consequences of granulophagy may be crucial to developing novel therapeutic strategies for these debilitating and often fatal conditions.
|Liu, Guangbo; Lanham, Clayton; Buchan, J Ross et al. (2017) High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots. PLoS One 12:e0174128|