Abstract 2189 A genome-wide screen identifies novel regulators of peroxisome biology

Eukaryotic cells are constantly adapting to their environment by remodeling their gene expression, proteome, and morphology. One fascinating example of cellular adaptation and versatility is the peroxisome, a membrane-bound organelle that is remodeled across evolution and development to meet the specific metabolic needs of cells. Study of the peroxisome has trailed that of other organelles such as the endoplasmic reticulum or the mitochondrion, perhaps because peroxisomes lack a single essential function. However, the versatility of the peroxisome is actually why it is so important to understand. Built on a chassis of a single membrane and a conserved suite of Pex proteins, peroxisomes help fungi make precursors to penicillin, help fireflies house luciferase to make light, and help oligodendrocytes make myelin to insulate neurons. A key gap in our understanding of human peroxisome homeostasis is our lack of a complete list of players. Past screens for PEX genes in mammalian cells have been thwarted by available genetic tools and the absence of a simple selection strategy for peroxisome function. To address these challenges, we engineered a CRISPRi cell line in which peroxisome import impacts cell survival by taking advantage of the peroxisome's selective permeability to the drug zeocin. By sequestering the zeocin resistance protein (ZeoR) in the peroxisome, we created a selection regime in which cells with functional peroxisomes are sensitive to zeocin because the peroxisomal ZeoR cannot neutralize the zeocin, but cells with impaired peroxisomes, and therefore cytosolic ZeoR, survive at high zeocin concentrations. Using this 'Pex-ZeoR' cell line, we then performed a genome-wide CRISPRi screen for guide RNAs that are enriched in zeocin selection conditions, finding gRNAs targeting known PEX genes as well as novel regulators impacting peroxisome biology, such as the E3 ligase RNF146. We will discuss our analysis of the hits from this screen, which reveal novel ways that peroxisome homeostasis integrates with cell signaling. This study was supported by a Connie Frank Fellowship to Jonathan Vu and the Searle Scholars and NIGMS R35 GM146784 award to Brooke Gardner.