Global protein synthesis is suppressed in hibernating C. elegans.

(A) A schematic representation of a typical cooling experiment. One day-old adult nematodes, grown at 20°C on multiple plates, were first adapted to cold at 10°C for 2 h, and then shifted to 4°C. At indicated time points, plates were transferred back to 20°C and the animals treated in an experiment-specific manner. Created with BioRender.com. (B) Polysome profiles from wild-type animals treated as shown in (A) and collected from 4°C at the indicated times. Marked are the positions of mono-, di-, and polysomes. Note a strong decrease of large polysomes with a concomitant increase of monosomes in cold, which become more pronounced with a longer cold exposure. (C) Protein synthesis evaluated with the SUnSET assay in animals incubated as indicated. Puromycin incorporation was detected on a western blot, with the corresponding quantification on the right. Actin (ACT-1) was used as a loading control. Error bars indicate the SEM of three biological replicates. Unpaired two-sided t-test was used for statistical analysis. ***: p < 0.001, ****: p < 0.0001.

Transcription is the main determinant of gene expression in the cold.

(A) Changes in mRNA abundance (mRNA, x-axis) and translation (RPF, y-axis) for all transcripts shifting wild-type animals from 10°C to 4°C. Each dot represents the log (base 2) fold change of a single transcript. The most upregulated transcript, lips-11, is indicated in green. Overall, note a strong correlation between mRNA levels and translation. A small subpopulation of transcripts (red) displayed little or no change in mRNA levels but showed reduced association with ribosomes, suggesting specific translation repression. (B) Top: Diagram representing a reporter construct, wherein GFP is expressed under the control of lips-11 promoter and unc-54 3’ UTR. Below are representative fluorescent micrographs, taken at the indicated conditions, of several bundled animals carrying the reporter GFP. The animals are outlined in the control panel and the positions of animals’ heads are indicated by asterisks. The scale bar = 200 µm.

Transcription of lips-11 is dependent on the IRE-1/XBP-1 pathway in the cold.

(A) Micrographs of several bundled animals expressing the Plips-11::GFP reporter, taken at the indicated conditions. Note a strong decrease in GFP reporter expression after one and three days at 4°C upon RNAi-mediated knockdown of ire-1, but not of atf-6 or pek-1. The animals are outlined in the mock control panel and the position of the head is indicated by asterisks for all animals. The scale bar = 200 µm. (B) Top: Representative micrographs of adult animals expressing the Pxbp-1::xbp-1::GFP splicing reporter at the indicated temperatures. GFP is expressed in frame only upon removal of the IRE-1 regulated intron in xbp-1 mRNA. Arrowheads indicate the outlined nucleus of the most anterior pair of intestinal cells. The scale bar = 40 µm. Below: the corresponding quantification of nuclear GFP reporter expression, relative to 20°C. Between two and five nuclei were analyzed per animal, in at least fifteen animals per condition. Error bars indicate the SEM. Unpaired two-sided t-test was used for statistical analysis. ****: p < 0.0001. (C) Micrographs of mock or xbp-1 RNAi treated Plips-11::GFP reporter animals, kept for one or three days at 4°C. The animals are outlined in the mock control panel and the position of the head is indicated by asterisks for all animals. The scale bar = 200 µm.

Cold specifically activates the UPRER through IRE-1.

(A) Micrographs of several bundled animals carrying the Phsp-4::GFP reporter, taken at the indicated temperature and time. The animals are outlined in the 20°C control panel for day 1 and the position of the head is indicated by asterisks for all animals. The scale bar = 200 µm.

(B) RT-qPCR analysis of fold change in mRNA levels for known UPRER target genes after three days at 4°C, relative to three days at 20°C. Dashed lines separate genes that are regulated by different pathways (IRE-1, ATF-6, and PEK-1). Note that the mRNA levels of all IRE-1 responsive genes are significantly upregulated at 4°C. Error bars indicate the SEM of three biological replicates. Unpaired two-sided t-test was used for statistical analysis. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Misfolded protein levels increase during short-term cold exposure.

Left: representative micrographs, taken at the indicated conditions, of several bundled animals carrying the CPL-1W32A,Y35A::YFP misfolding reporter. The head of each animal is indicated by pharyngeal expression of mCherry, driven by the myo-2 promoter. Animals are outlined in the 20°C control panel for day 1. The scale bar = 200 µm. Right: the corresponding quantification of YFP fluorescence at 4°C after one or three days, relative to 20°C. YFP fluorescence was analyzed from whole animals, with a minimum of 38 animals per condition. Error bars indicate the SEM. Unpaired two-sided t-test was used for statistical analysis. ns: p > 0.05, ****: p < 0.0001.

Choline supplementation suppresses IRE-1 regulated gene expression in the cold.

(A) Left: Micrographs taken after one or three days at 4°C of several bundled Plips-11::GFP reporter animals on a mock of 50 mM choline supplemented diet. Mock treated animals are outlined in the 4°C control panel for day 1 and the head of all separate animals are indicated with asterisks. The scale bar = 200 µm. Right: the corresponding quantification of intestinal GFP fluorescence after one or three days at 4°C on a choline supplemented diet, relative to a mock diet. GFP fluorescence was analyzed from a minimum of 38 animals per condition. Error bars indicate the SEM. Unpaired two-sided t-test was used for statistical analysis. *: p < 0.05, ****: p < 0.0001. (B) Left: Micrographs taken after one or three days at 4°C of several bundled Phsp-4::GFP reporter animals on a mock of 50 mM choline supplemented diet. Mock treated animals are outlined in the 4°C control panel for day 1 and the head of all separate animals are indicated with asterisks. The scale bar = 200 µm. Right: the corresponding quantification of intestinal GFP fluorescence after one or three days at 4°C on a choline supplemented diet, relative to a mock diet. GFP fluorescence was analyzed from a minimum of 28 animals per condition. Error bars indicate the SEM. Unpaired two-sided t-test was used for statistical analysis. ****: p < 0.0001.

IRE-1 facilitates cold survival in C. elegans during extreme hypothermia.

(A) Survival curves of wild-type and ire-1(ok799) mutant animals at 4°C. Error bars indicate the SEM from 6 biological replicates. A minimum of 800 animals were scored per time point. Wilcoxon signed-rank test was used for statistical analysis; p = 0.03. (B) Graphical model of IRE-1 activation in the cold. Cold exposure leads to increased protein misfolding and lipid disequilibrium in the ER. These two stressors trigger the activation of IRE-1, which promotes the downstream processing of xbp-1u to xbp-1s mRNA. The functional XBP-1 transcription factor enters the nucleus to promote transcription of specific genes, including those facilitating cold survival. Created with BioRender.com.

(A) PCA plot of ribosome profiling samples and replicates based on transcript RFP FPKM. Note co-clustering of replicates and temperatures. (B) Changes in translation for selected genes as a function of temperature changes and exposure.

Micrographs taken of several bundled Plips-11::GFP reporter animals after 6 hours of treatment with DMSO or tunicamycin. DMSO treated control animals are outlined in the left panel and the head of all separate animals are indicated with asterisks. The scale bar = 200 µm.

(A) Micrographs taken of several bundled Phsp-6::GFP UPRmito reporter animals, taken at the indicated time and temperature. Animals are outlined in the 20°C control panel for day 1 and the head of all separate animals are indicated with asterisks. The scale bar = 200 µm. (B) Micrographs taken of several bundled Phsp-60::GFP UPRmito reporter animals, taken at the indicated time and temperature. Animals are outlined in the 20°C control panel for day 1 and the head of all separate animals are indicated with asterisks. The scale bar = 200 µm. (C) RT- qPCR analysis of fold change in mRNA levels for known UPRER target genes after one day at 4°C, relative to one day at 20°C. Dashed lines separate genes that are regulated by different pathways (IRE-1, ATF-6, and PEK-1). Error bars indicate the SEM of three biological replicates. Unpaired two-sided t-test was used for statistical analysis. ns: p > 0.05, *: p < 0.05.

Left: Micrographs taken after one or three days at 4°C of several bundled Plips-11::GFP reporter animals on a 0.1% Tergitol mock diet or a 0.8 mM oleic acid, 0.8 mM palmitic acid, or 0.8 mM linoleic acid supplemented diet. Mock treated animals after one day at 4°C are outlined and the head of all separate animals are indicated with asterisks. The scale bar = 200 µm. Right: the corresponding quantification of intestinal GFP fluorescence after one or three days at 4°C on the different fatty acid supplemented diets, relative to the mock diet. GFP fluorescence was analyzed from 21 to 31 animals per condition. Error bars indicate the SEM. Unpaired two-sided t-test was used for statistical analysis. N.s.: p > 0.05, ***: p < 0.001, ****: p < 0.0001.

Four-way Venn diagram depicting the overlap between genes with a minimum 2-fold enrichment (10°C to 4°C day 1) and previously reported IRE-1 responsive genes that are upregulated either during the UPRPT (Shen et al., 2005), upon shifting animals from 15°C to 2°C (Dudkevich et al., 2022), or during the UPRLBS (Koh et al., 2018).