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. 2018 Apr 17;7:e35037. doi: 10.7554/eLife.35037

Figure 3. ZIP-10 acts downstream of ISY-1 and mediates transcriptional response to CW.

(A) QPCR measurements of asp-17 levels induced by CW in wild type and isy-1(dma50) mutants. (B) Volcano plot of RNA-seq showing differentially regulated genes (up-regulated genes in red; down-regulated genes in green) in isy-1 mutants compared with wild type. (C) RNA-seq measurements of expression levels for indicated genes in wild type and isy-1(dma50) mutants. (D) Nomarski and fluorescence images showing asp-17p::GFP induction by isy-1 RNAi was blocked in zip-10 mutants. (E) Western blots of the integrated zip-10p::zip-10::EGFP::FLAG strain showing time-dependent protein induction by CW. (F) Western blots of the integrated zip-10p::zip-10::EGFP::FLAG strain showing its up-regulation by isy-1 RNAi and down-regulation by GFP or zip-10 RNAi. Both short- and long-exposure blots are shown. (G) Western blots of the integrated zip-10p::zip-10::EGFP::FLAG strain showing its up-regulation strictly required warming after cold shock. (H) Western blots of the integrated zip-10p::zip-10::EGFP::FLAG strain showing its up-regulation by CW was further enhanced by isy-1 RNAi. (I) QPCR measurements of gene expression levels showing ZIP-10 dependent up-regulation of asp-17 and cpr-3 but not srr-6 or F53B9.1 after cold for indicated durations and 1 hr warming. n ≥ 20 total animals for each group with N ≥ 3 independent biological replicates; *** indicates p<0.001. Scale bar: 20 µm.

Figure 3—source data 1. Lists of genes up- and down-regulated by the isy-1(dma50) mutation with adjusted p<0.05 and log2FoldChange from biological triplicate samples of wild-type and isy-1(dma50) mutant C. elegans.
elife-35037-fig3-data1.xlsx (289.6KB, xlsx)
DOI: 10.7554/eLife.35037.012

Figure 3.

Figure 3—figure supplement 1. ISY-1 does not affect general splicing of a GFP reporter nor specific splicing of zip-10.

Figure 3—figure supplement 1.

(A) Nomarski and fluorescence images showing that animals with isy-1 RNAi are not defective in general intron splicing of an eft-3p::GFP (intron-containing) reporter. (B) Read plot from RNA-seq of wild type at the zip-10 locus. (C) Read plot from RNA-seq of isy-1(dma50) mutants at the zip-10 locus indicating the intact splicing of the zip-10 intron despite increased mRNA levels. (D) Normalized QPCR measurements of mRNA expression levels of isy-1 and zip-10 from wild type and isy-1(dma50) mutants. (E) Normalized QPCR measurements of mRNA expression levels of isy-1 and zip-10 from wild type and CW-treated animals.

Figure 3—figure supplement 2. Mechanisms of regulation and function of zip-10.

Figure 3—figure supplement 2.

(A) Nomarski and fluorescence images showing the zip-10p::GFP reporter activated by isy-1 RNAi. (B) Western blot of lysates from zip-10p::zip-10::EGFP::FLAG transgenic animals showing its increased abundance after CW but not by hypoxia (0.5% O2) or starvation for 24 hrs. (C) Western blot of fractionated lysates from zip-10p::zip-10::EGFP::FLAG transgenic animals after CW showing its increased abundance in cytosol and nucleus. (D) Western blot of lysates from zip-10p::zip-10::EGFP::FLAG transgenic animals showing its increased abundance after CW when warming was permitted and cold exposure was prolonged. (E) Western blot of lysates from zip-10p::zip-10::FLAG transgenic and various RNAi-treated animals showing its increased abundance after CW in isy-1 RNAi-treated animals but was unaffected by RNAi against C37C3.2, encoding the C. elegans orthologue of translation initiation factor 5 (eIF5), or T26A8.4, encoding the C. elegans orthologue of Saccharomyces cerevisiae Caf120, a component of the Ccr4-Not deadenylase RNA-degrading complex. (F) ZIP-10 domain organization based on analysis by SMART (smart.embl-heidelberg.de) and modeled structure of ZIP-10’s bZIP domain (swissmodel.expasy.org). (G) Sequence alignment of ZIP-10’s bZIP domain and the Maf transcription factor as a template of the modelled ZIP-10 structure. (H) Schematic of the zip-10 locus showing the wild type, EGFP::FLAG tagged allele, and the wild-type and C-terminal transactivation mutant ZIP-10 protein sequences. The mutated residues (from Q and N to V and A, respectively) at the ZIP-10 C-terminus are completely conserved in nematodes.