Unfolding the unfolded protein response: Turn to the worm!

Abstract

The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated upon endoplasmic reticulum (ER) stress, which is functionally defined as the imbalance between a protein folding load facing the ER and the organelle's ability to process that load. In mammals, the UPR contributes to adaptation as well as pathogenesis to diverse disease states. To dissect UPR-signaling in a multicellular organism, we have used <italic>C. elegans</italic> as a genetic model and identified its homologues of the three mammalian ER stress signaling molecules, which are termed as <italic>ire-1</italic>, <italic>pek-1</italic> and <italic> atf-6</italic>. Mammalian homologues of IRE-1 and ATF-6 upregulate transcription of genes required for protein folding and ER-associated degradation, while a major function of mammalian PEK-1 is to attenuate translation to decrease the ER folding load. I identified the <italic>xbp-1</italic> mRNA as an IRE-1 splicing substrate. IRE-1 mediated cleavage of <italic>xbp-1</italic> mRNA upon ER stress removes a 23-nucleotide intron that is required to activate UPR gene transcription. Both loss-of-function worms in <italic>ire-1</italic> and <italic>pek-1</italic> genes were hypersensitive to treatments inducing ER stress, suggesting <italic> ire-1</italic> and <italic>pek-1</italic> are required for survival to ER stress. Genetic deletion of both <italic>ire-1</italic> and <italic>atf-6 </italic> genes caused worms to arrest at a young larval stage with an intestinal defect similar to that observed in <italic>ire-1</italic>; <italic>pek-1</italic> double mutants, suggesting that <italic>ire-1/xbp-1</italic> acts with either <italic> pek-1</italic> or <italic>atf-6</italic> in complementary pathways that are essential for larval development. Therefore, I propose that the UPR mediated by <italic>ire-1</italic>, <italic>pek-1</italic> and <italic>atf-6</italic> maintain ER homeostasis allowing proper development. The results also suggest that <italic>atf-6</italic> and <italic>pek-1</italic> could function genetically in the same pathway, or mediate independent signaling cascades on ER stress. Interestingly, <italic>atf-6</italic> and <italic>pek-1</italic> double mutants did not display an obvious phenotype, supporting that <italic>ire-1</italic> signaling is sufficient to provide UPR adaptive functions, ensuring proper growth and differentiation. To elucidate how <italic>atf-6</italic> and <italic> pek-1</italic> are interrelated, I am performing a genetic screen in attempt to identify new molecules regulating the UPR. This screen combines classic chemical mutagenesis and recently developed bacterial feeding-mediated RNA interference techniques. I screened for mutations that cause synthetic lethality with loss of either <italic>ire-1</italic> or <italic>pek-1</italic>. This screen identified a missense mutation in <italic>ire-1</italic> that caused a loss of function phenotype, demonstrating the feasibility of this strategy. In summary, we established a model system---<italic>C. elegans</italic>---to study the UPR in metazoan species and identified the genetic interactions among the key regulatory molecules that respond to ER stress. Identification of new components in the UPR by our ongoing genetic screen and microarray analysis should reveal additional functions of the UPR and shed light on the mechanism by which cells coordinate transcriptional and translational regulation to adapt to the increased protein-folding demand.