UBC Theses and Dissertations

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UBC Theses and Dissertations

Identification of metabolic alterations that activate the unfolded protein response of the endoplasmic reticulum in C. elegans Xu, Jiaming


Endoplasmic reticulum (ER) stress due to protein misfolding or membrane lipid imbalance is observed in many diseases. ER homeostasis can be restored by activation of the unfolded protein response (UPR-ER), which, in higher eukaryotes, consists of three parallel 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. These sensors activate downstream effectors to restore cellular homeostasis. However, we lack a global view of genetic perturbations that activate the UPR-ER in metazoans. To identify\metabolic pathways that affect ER homeostasis, I used RNA interference (RNAi) to inactivate 1247 metabolic genes in Caenorhabditis elegans using an IRE-1 branch specific transcriptional reporter, hsp-4p::gfp. After screening and validation, I obtained 34 high-confidence hits that also activate the PEK-1 branch. Next, using a strain lacking the key IRE-1 pathway effector XBP-1 (xbp-1; hsp-4p::gfp), I showed that these gene inactivations induce canonical IRE-1 signaling. Moreover, dietary choline supplementation, which suppresses UPR-ER in worms defective for phosphatidylcholine (PC) synthesis pathway, partially suppresses UPR-ER activation in 3 of the 34 hits, suggesting that most hits do not activate the UPR-ER via defective PC synthesis. Finally, I performed follow-up studies on two of the 34 hits, the primases pri-1 and pri-2, whose inactivation causes DNA damage due to replication fork stalling. These two RNAi clones were selected because both activate hsp-4p::gfp in C. elegans embryos in a partially ire-1-, xbp-1-independent manner. I observed that pri-1 and pri-2 RNAi specifically induce the UPR-ER, but not the mechanistically distinct cytosolic or mitochondrial UPRs. This suggests that pri-1 and pri-2 RNAi do not cause global protein misfolding. Interestingly, genomic instability caused by loss of DNA repair pathways did not activate the UPR-ER, suggesting that replication stress specifically activates 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 mechanisms of UPR-ER regulation. Furthermore, as many genes identified here have human homologues, my data may provide a starting point for the discovery of novel drug targets for human diseases featuring ER dysfunction.

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