Combining Auxin-Induced Degradation and RNAi Screening Identifies Novel Genes Involved in Lipid Bilayer Stress Sensing in Caenorhabditis elegans

Abstract

Alteration of the lipid composition of biological membranes interferes with their function and can cause tissue damage by triggering apoptosis. Upon lipid bilayer stress, the endoplasmic reticulum mounts a stress response similar to the unfolded protein response. However, only a few genes are known to regulate lipid bilayer stress. We performed a suppressor screen that combined the auxin-inducible degradation (AID) system with conventional RNAi in C. elegans to identify members of the lipid bilayer stress response. AID-mediated degradation of the mediator MDT-15, a protein required for the upregulation of fatty acid desaturases, induced the activation of lipid bilayer stress-sensitive reporters. We screened through most C. elegans kinases and transcription factors by feeding RNAi. We discovered nine genes that suppressed the lipid bilayer stress response in C. elegans. These suppressor genes included drl-1/MAP3K3, gsk-3/GSK3, let-607/CREB3, ire-1/IRE1, and skn-1/NRF1,2,3. Our candidate suppressor genes suggest a network of transcription factors and the integration of multiple tissues for a centralized lipotoxicity response in the intestine. Thus, we demonstrated proof-of-concept for combining AID and RNAi as a new screening strategy and identified eight conserved genes that had not previously been implicated in the lipid bilayer stress response.

ATF-4 and hydrogen sulfide signalling mediate longevity from inhibition of translation or mTORC1

“…Mutant strains used are described at www.wormbase.org: LGI: eif-2a(qd338); LGII: cth-2(mg599), raga-1(ok386), spe-9(hc88), rrf-3(pk1426 and b26), eat-2(ad1116), rict-1(ft7) ; LGIII: rars-1(gc47), daf-2(e1368, e1370), glp-1(e2141), clk-1(qm30) ; LGX: atf-4(tm4397, tm4212) . Transgenic strains: LD1499 [P atf-4 (uORF)::GFP:: unc-54 (3’UTR)] was made by Chi Yun (1.8kb promoter 5’ of atf-4 including both uORFs into pPD95.75, personal communication with Chi Yun and David Ron) 63 .…”

Beyond Proteostasis: Lipid Metabolism as a New Player in ER Homeostasis

“…In C. elegans, the functional mapping and characterization of genetic pathways is typically done by identifying mutants with a desired phenotype (e.g., activation of a reporter gene, synthetic lethality with another gene, increased sensitivity to a chemical, etc.). These classical genetic approaches have been extremely useful in pathway mapping and have helped identify novel lipid metabolism regulators of the UPR-ER [13,14]. For example, recent efforts have used functional genomics approaches to identify new players in the UPR-ER LBS of C. elegans.…”

“…Specifically, a reverse genetic screen depleting 1247 predicted metabolic genes by RNAi yielded 34 genes whose inactivation induces the UPR-ER, including previously known players in lipid homeostasis and new candidates whose specific links to UPR-ER have not been explored [13]. Another study screened 712 kinase and transcription factor genes and identified 8 genes whose inactivation suppresses the UPR-ER LBS (induced by fat-6, mdt-15, or sams-1 RNAi) but not the UPR-ER PT (induced by heat or tunicamycin) [14]. This is exciting as it provides a list of new genes that may specifically activate the UPR-ER LBS or may selectively be required for a functional UPR-ER LBS , respectively.…”

“…Pertinent to the study of the UPR-ER, these include (i) a relative lack of accessibility to manipulation with drugs, which sometimes fail or work only at extremely high doses (e.g., chemical chaperones, see below); (ii) differences in membrane lipid composition, as C. elegans features very low to no cholesterol in its cellular and subcellular membranes; and (iii) mechanistic differences concerning nuanced roles of the UPR-ER, such as regulated IRE1-dependent decay of mRNA (RIDD; see below). Nevertheless, because of the high level of conservation of the core UPR-ER pathways and of (lipid) metabolism, and because of the powerful genetic and genomic tools including forward and reverse genome-wide screens, C. elegans is an excellent model that has provided important new insights into the mechanistic basis of UPR-ER regulation under different stress conditions, including lipotoxicity [13][14][15][16][17][18][19][20][21][22].…”

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences

“…Expression of ATF-4 can be increased in response to endoplasmic reticulum (ER) stress through PERK1/PEK-1 phosphorylation of the initiation factor eIF2α, which reduces global translation rates [ 63 ]. PERK1/PEK-1 activation is also a hallmark of the ER unfolded protein response (UPR ER ) [ 64 ].…”