Endoplasmic reticulum (ER) stress due to protein misfolding is observed in many diseases such as cancer, diabetes, and metabolic diseases. ER homeostasis can be restored by activation of the unfolded protein response (UPR-ER), which alleviates the molecular stress or induces cell death when damage is too severe. In higher eukaryotes, the UPR consists of three branches: The Inositol-Requiring-Enzyme 1 (IRE-1) branch, the protein kinase RNA-like ER kinase (PEK-1) branch, and the Activating Transcription Factor 6 (ATF-6) branch. When misfolded proteins accumulate in the ER lumen, these sensors activate downstream effectors, which together attenuate global translation and transcriptionally upregulate genes that restore homeostasis. Interestingly, abnormal membrane lipid composition also induces the UPR-ER, independent of protein misfolding. However, to date we lack a global view of genetic perturbations that activate the UPR-ER in metazoans. To identify proteotoxicity-independent metabolic pathways that affect ER homeostasis, I used RNA interference (RNAi) to inactivate 1247 metabolic genes in Caenorhabditis elegans with the IRE-1 branch specific transcriptional reporter,
hsp-4p::gfp. After screening and rigorous validation, I obtained 34 high-confidence
xbp-1-dependent hits that also activate the PEK-1 branch. Next, I used quantitative real-time PCR to show that 11 of 15 tested RNAi clones induce the endogenous UPR-ER in wild-type worms. Then I tested whether dietary choline supplementation, which suppresses UPR-ER in worms defective for phosphatidylcholine (PC) synthesis pathway, is sufficient to suppress UPR-ER activation in our hits. Of the 34 hits, 3 were partially rescued by dietary supplementation of choline along with the complete rescue of
sams-1 RNAi-treated animals, suggesting majority of the hits does not activate the UPR-ER via defective PC synthesis. Finally, I performed follow-up studies on one of the hit pathways on DNA replication. In early embryos, DNA replication stress induces UPR-ER activation, but not the mechanistically distinct cytosolic or mitochondrial UPRs, in a partially
ire-1-,
xbp-1-independent manner. This suggests that replication stress does not trigger global protein misfolding. Interestingly, genomic instability caused by loss of DNA repair pathways such as mismatch repair, nucleotide excision repair, and cohesin, which is essential for proper chromosome segregation, did not activate the UPR-ER, suggesting that molecular events specific to replication stress activate
hsp-4 and the UPR-ER in the embryo. In sum, by identifying new genes that affect UPR-ER homeostasis in C. elegans, my project provides new insights into UPR-ER regulation and may serve as a starting point for the discovery of drug targets for human diseases featuring UPR-ER dysfunction.