Introduction

One constant facet of hibernation appears to be the global reduction of protein synthesis. It is manifested, among others, by the loss of polyribosomes (aka polysomes associated with robust translation) and the inhibition of basic translation factors like eIF2α and eEF2 that are required for the initiation and elongation steps of translation, respectively (Chen et al., 2001; Frerichs et al., 1998; Knight et al., 2000; Van Breukelen and Martin, 2001). Cells from non-hibernating species, including humans, also respond to cold by downregulating translation (Hofmann et al., 2012). At the same time, numerous studies highlight the importance of cold-induced gene expression, raising questions about the mechanism facilitating the expression of specific genes against the overall reduction of protein synthesis.

Hibernation has been traditionally studied in non-standard animal models like squirrels, bats, or bears. Nonetheless, simple genetic models are often advantageous in dissecting complex biological phenomena. Thus, to better understand gene expression underlying cold adaptation, we employ the nematode C. elegans: a rapid, morphologically simple, and genetically tractable animal model. These animals thrive in temperate climates, indicating that, in the wild, they can tolerate spells of cold (Frezal and Felix, 2015). In a laboratory, deep cooling of C. elegans either leads to death (when the cooling is rapid) (Habacher et al., 2016; Ohta et al., 2014; Robinson and Powell, 2016) or to a dormant hibernation-like state (when the cooling is gradual) (Habacher et al., 2016; Ohta et al., 2014). During “hibernation” C. elegans suppresses aging (Habacher et al., 2016). Although this response and mammalian hibernation are not equivalent, some aspects appear to be conserved (Pekec et al., 2022). Thus, the advantages of a simple genetic model may be useful in uncovering conserved aspects of cellular adaptations to cold.

Here, we demonstrate that C. elegans, like hibernating animals, respond to cold by globally reducing mRNA translation. Overall, the translation of individual transcripts correlates with their levels, suggesting that cold-specific gene expression is regulated primarily through transcription. We validated this hypothesis by focusing on one up-regulated gene, lips-11, which encodes a putative lipase implicated in unfolded protein response (UPR) (Shen et al., 2005). The UPR consists of three stress-sensing and transducing branches: IRE1, PEK1, and ATF6, all conserved in C. elegans (Hetz et al., 2020). Once activated, the UPR restores homeostasis by various means, including by remodeling transcription and translation. We find that, in C. elegans, cold activates the IRE-1 branch of the UPR. While IRE-1 signaling is induced typically in response to protein misfolding, we present evidence that, in the cold, the IRE1 pathway responds to both protein and lipid bilayer stress in the endoplasmic reticulum (ER) and that its activation is beneficial for hibernating animals.

Results

Protein synthesis is globally reduced in hibernating C. elegans

In several species and cultured cells, cooling leads to a global reduction of translation (Chen et al., 2001; Frerichs et al., 1998; Hofmann et al., 2012; Knight et al., 2000; Van Breukelen and Martin, 2001). To test if this occurs in C. elegans, we used a previously described cooling paradigm and sampling across different temperatures and time points (Figure 1A). We combined this with either polysome profiling or SUrface SEnsing of Translation (SUnSET). The former method separates translated mRNAs according to the number of bound ribosomes. The latter involves the incorporation of puromycin into newly synthesized peptides and subsequent detection of the incorporated puromycin by western blotting (Arnold et al., 2014; Schmidt et al., 2009). By polysome profiling, we observed in the cold a massive loss of polyribosomes (multiple ribosomes associated with mRNAs), with a concomitant increase in monosomes (Figure 1B). By SUnSET, we observed a stark drop in puromycin incorporation in hibernating animals (Figure 1C). Thus, consistent with observations in other species, severe cooling is accompanied in C. elegans by a global reduction of protein synthesis.

Figure 1.

Transcription is a major determinant of cold-specific gene expression

Although global translation decreases in the cold, the translation of specific transcripts still could be regulated, i.e., activated or repressed. To examine this possibility, we combined ribosomal profiling with total RNA sequencing across different temperatures and time points (Supplementary files 1 and 2). We made several observations. First, while shifting animals from 20°C to 10°C had a relatively mild impact on translation (ribosomal occupancy), the subsequent shift from 10°C to 4°C had a strong effect. Then, continuing the incubation at 4°C again had a relatively small effect (Figure 2A and Figure 2A – figure supplement 1). Thus, most translational remodeling occurs during the first day of incubation at 4°C. Focusing on this transition (10°C to 4°C day 1), we observed a good correlation between mRNA levels and translation (Figure 2A). Thus, although general protein synthesis is reduced in the cold, the available mRNAs appear to be translated. However, we did observe some exceptions, such as a group of transcripts whose translation appeared reduced with no concomitant drop in mRNA levels (red dots in Figure 2A), suggesting the existence of dedicated repressive mechanism(s). Intriguingly, this group includes mRNAs encoding three fatty acid desaturases (FAT-2, -3, and -4; Supplementary file 3). This finding is surprising, because desaturation of membrane lipids has been thought to play an important role in cold adaptation (Hayward et al., 2007).

Figure 2.

For most transcripts, however, their translation correlates with their levels. In other words, the abundance of a particular protein appears to depend primarily on the abundance of its mRNA. Since transcription is the main determinant of mRNA levels, these results suggest that cold-specific gene expression primarily depends on transcription activation. To test this, we selected one gene, lips-11, whose expression increased the most during the shift from 10°C to 4°C (green dot in Figure 2A). To test if cold induces lips-11 transcription, we generated a strain expressing a GFP reporter, whose expression depends on the endogenous lips-11 promoter (Plips-11) and the unc-54 3’UTR (permitting unregulated expression of the attached open reading frame). We observed that the lips-11 promoter was sufficient to induce GFP expression in the cold (Figure 2B). Thus, while we cannot exclude that cold additionally impacts the stability of some mRNAs, we propose that cold-specific gene expression is shaped mainly at the transcription level.

Cold-induced transcription of lips-11 depends on the IRE-1/XBP-1 branch of the UPR

Intriguingly, lips-11 belongs to genes activated by the UPR during ER stress (UPR^ER) (Shen et al., 2005). Indeed, we observed that the Plips-11 GFP reporter’s levels increased upon treating animals with the protein glycosylation inhibitor tunicamycin, a UPR^ER inducer (Figure 3 – figure supplement 1). This observation prompted us to examine if the induction of lips-11 in the cold depends on a particular branch of the UPR^ER signaling; IRE-1, ATF-6, or PEK-1. Indeed, we observed reduced levels of the lips-11 reporter upon RNAi-mediated depletion of ire-1, but not atf-6 or pek-1 (Figure 3A). Thus, lips-11 expression in the cold depends on the UPR^ER signal transducer IRE-1.

Figure 3.

In response to ER stress, the endoribonuclease domain of IRE-1 is activated and promotes the processing of xbp-1 mRNA, which gives rise to the functional form of the XBP- 1 transcription factor. As XBP-1-independent functions of IRE-1 have been reported, we first examined if xbp-1 mRNA is processed in the cold. To do this, we used a published xbp-1 splicing reporter strain, wherein the xbp-1 promoter drives the expression of genomic xbp-1 fused to GFP, which is expressed in frame upon processing by activated IRE-1 (Ozbey et al., 2020). Importantly, we observed a significant upregulation of XBP-1::GFP in the cold (Figure 3B), indicating the activation of the IRE-1/XBP-1 branch of the UPR^ER. Moreover, in addition to IRE-1, the cold-induced expression of the lips-11 reporter also depended on XBP-1 (Figure 3C). Thus, the upregulation of lips-11 in hibernating C. elegans depends on the IRE-1/XBP-1 branch of the UPR^ER.

Hibernation specifically activates the IRE-1 branch of the UPR^ER

The activation of IRE-1 in the cold suggests that hibernating animals experience ER stress. To confirm this, we utilized a commonly used UPR^ER reporter strain, wherein GFP expression is driven by the promoter of hsp-4, the C. elegans homolog of the ER chaperone BiP (Calfon et al., 2002). Indeed, the expression of this reporter gradually increased in the cold (Figure 4A). By following the expression of mitochondrial chaperones hsp-6 (homolog of HSP70) and hsp- 60 (homolog of HSP60), we additionally examined if cold elicits UPR in the mitochondria (UPR^mito) (Yoneda et al., 2004). However, by contrast to hsp-4, neither hsp-6 nor hsp-60 were induced in the cold (Figure 4A – figure supplement 1). Thus, hibernating animals appear to experience stress in the ER but not mitochondria.

Figure 4.

While we showed that lips-11 upregulation is mediated by the IRE-1/XBP-1 pathway, other branches of the UPR^ER could be activated in the cold. Testing this possibility, we examined the expression of select UPR^ER target genes regulated through the IRE-1 (dnj-27, srp-7, C36B7.6), the ATF-6 (cht-1, ZC168.2), or the PEK-1 (cbp-3, R02D3.8) pathway (Shen et al., 2005). While the levels of these transcripts did not change significantly after one day in the cold (Figure 4B – figure supplement 1), four transcripts were more abundant after three days (Figure 4B). Notably, 3/4 of them are known targets of the IRE-1 pathway. Although a more comprehensive analysis is needed to reach firm conclusions, these findings suggest that cold triggers the UPR^ER primarily via the activation of IRE-1.

The cold-induced UPR^ER stems from both protein misfolding and lipid disequilibrium

ER stress and subsequent activation of the UPR^ER are typically associated with misfolded proteins accumulating in the ER lumen. To test if cold aggravates protein misfolding, we employed another reporter strain, wherein YFP is fused to a mutated form of the C. elegans cathepsin L-like protease (CPL-1^W32A,Y35A), which does not fold properly and accumulates during ER stress (Efstathiou et al., 2022). We found that the levels of misfolded CPL-1 modestly increased after one day in the cold but then returned to basal levels after a longer cold exposure (Figure 5). Thus, disturbed ER proteostasis could, at least transiently, trigger the activation of UPR^ER in hibernating animals.

Figure 5.

Apart from protein misfolding, lipid disequilibrium is also known to trigger UPR^ER (Halbleib et al., 2017; Tam et al., 2018; Volmer et al., 2013). The IRE-1 pathway is specifically activated when the ratio between phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreases, or when the levels of unsaturated fatty acids become insufficient (Ariyama et al., 2010; Hou et al., 2014; Thibault et al., 2012). In the latter case, reduced content of unsaturated fatty acids (FAs) results in a decreased ER membrane fluidity, which is thought to increase the oligomerization and thus the activation of IRE-1 (Halbleib et al., 2017). So, we asked next if dietary supplementation of unsaturated FAs or choline (crucial for PC synthesis) impacts the IRE-1-dependent induction of the lips-11 reporter in the cold. We found that supplementing the diet with either saturated (palmitic acid) or unsaturated FAs (oleic acid or linoleic acid) did not prevent the expression of the lips-11 reporter (Figure 6 – figure supplement 1; if anything, adding oleic acid appeared to upregulate the reporter). While these experiments have certain caveats (for example it is impossible to precisely control the concentration of internalized FAs), they suggest that insufficient lipid desaturation is not the main trigger of UPR^ER in the cold. By contrast, the dietary supplementation of choline led to reduced expression of lips-11 and hsp-4 reporters in hibernating animals (Figure 6A and Figure 6B, respectively). Together, these experiments suggest that cold-induced activation of UPR^ER could be triggered by sensing disruptions in both protein and lipid homeostasis, with the latter related to insufficient levels of PC rather than unsaturated FAs.

Figure 6.

IRE-1-dependent signaling is important for cold survival

Cold triggers the IRE-1/XBP-1 signaling pathway, but for what purpose? If it helps animals in cold adaptation, then inhibiting this pathway may be expected to impair cold survival. At standard temperature, the loss-of-function ire-1(ok799) mutants display no obvious defects. In the cold, however, we observed that these mutants survived less well than the wild type (Figure 7A). Thus, IRE-1/XBP-1 signaling appears to improve cold survival, presumably by activating its downstream target(s). A natural candidate is LIPS-11, but we found that its RNAi-mediated depletion had no obvious impact on cold survival. Assuming that the RNAi was effective, it suggests that either LIPS-11 plays no role in cold survival, or it functions redundantly with other IRE-1/XBP-1 targets. Several previous studies reported IRE-1-dependent genes activated in response to protein misfolding (UPR^PT; (Shen et al., 2005), lipid bilayer stress (UPR^LBS; (Koh et al., 2018), or cold when using different cooling conditions (Dudkevich et al., 2022). To examine if these genes are also activated by cold treatment in this study, we extracted them from the publications (Supplementary file 4) and examined their overlap with the cold- induced transcripts from Figure 2A. Curiously, we observed relatively little overlap between the different gene sets (Figure 7 – figure supplement 1). While the UPR^PT and the UPR^LBS are known to regulate largely distinct targets (Koh et al., 2018), this analysis further suggests than IRE-1-mediated gene activation is largely context-dependent. Also, since many cold- induced genes from this study appear to be unrelated to the examined IRE-1 gene sets, we hypothesize that additional pathways(s) work alongside the IRE1/XBP-1 pathway to shape gene expression in the cold.

Figure 7.

Summarizing our findings in a model (Figure 7B), cold-induced protein and lipid stress in the ER specifically induces the IRE-1 branch of the UPR. Following the IRE-1-mediated processing of xbp-1 mRNA, the XBP-1 transcription factor enters the nucleus and activates its target genes. These include lips-11, but the regulation of additional targets is necessary to explain the beneficial effect of IRE-1 on cold survival.

Discussion

Our findings indicate that C. elegans respond to severe cold by decreasing protein synthesis, consistent with observations in other models. Since translation is one of the most energy- consuming biological processes, its global reduction will help preserve cellular energy stores. In poikilotherms (animals whose body temperature depends on the environment), reducing global translation is part of their physiological response to cold. A similar situation occurs in homeotherms (animals whose temperature depends on metabolism) who are capable of temporal heterothermy, i.e., can change their body temperature in a circadian or seasonal circle. In other homeotherms (like humans), the global reduction of protein synthesis may help survive accidental hypothermia or repair peripheral cold injuries affecting the extremities and exposed skin.

Despite the general reduction of protein synthesis in the cold, most mRNAs continue to be translated, albeit at a lower rate. Consequently, either transcription or mRNA stability could regulate cold-specific gene expression. We present evidence that the expression of some cold-inducible genes depends on the transcription factor XBP-1, which functions downstream from IRE-1. Curiously, another cooling procedure (involving shifting animals from 15°C to 2°C) was reported to elicit IRE-1 signaling independently from XBP-1. In that case, IRE-1 activation occurs in neurons, but it stimulates the remodeling of lipid metabolism in other tissues (Dudkevich et al., 2022). In that context, IRE-1 was proposed to adjust the balance between saturated and unsaturated FAs, because supplementing the diet with unsaturated FAs could bypass the requirement for IRE-1. By contrast, the IRE-1 signaling reported here depends on XBP-1, and the dietary supplementation of non-saturated FAs did not prevent the activation of IRE-1/XBP-1 signaling. This observation is consistent with a previous study, which showed that inhibiting C. elegans fatty acid desaturases has relatively little impact on C. elegans tolerance of severe cold (Murray et al., 2007). Combined with our observation that some FA desaturases are translationally repressed in the cold, these findings collectively suggest that desaturated FAs are not limiting in hibernating C. elegans, although the situation appears to be different when these animals are cultured at low but physiologically tolerable temperatures (Svensk et al., 2013). Summarizing, cold seems to activate IRE-1-mediated signaling in various ways and with different outcomes, possibly influenced by the animal’s physiological state and specific cooling conditions.

What triggers the activation of the IRE-1/XBP-1 pathway in the cold? Using the CPL- 1^{W32A, Y35A} protein folding sensor, we observed a transient misfolding (present at day 1, but not 3 in the cold). Since protein synthesis is strongly downregulated in the cold, the burden of misfolded proteins may decrease over time, suggesting that additional cues evoke UPR^ER at later stages of hibernation. Intriguingly, the dietary supplementation of choline reduced IRE- 1-dependent expression during both early and later stages of hibernation, suggesting that alterations involving one or more choline derivatives may contribute to ER stress. Since choline is essential for PC synthesis (a critical component of biological membranes (Akkers et al., 2010)), the IRE-1 pathway may monitor a PC-sensitive aspect of ER biology. For example, it could detect alterations in transmembrane channels or peripheral membrane-binding proteins, whose activities depend on the physical properties of membrane lipids (Allende et al., 2004; Janmey and Kinnunen, 2006).

Finally, while the activation of IRE-1-dependent signaling appears to be an important aspect of C. elegans responses to cold, is it also used in other animals? There is emerging evidence suggesting that it could be so. In culture, human neurons exposed to moderate hypothermia activate the ER stress response and induce all three branches of the UPR, including the IRE1 (Rzechorzek et al., 2016). Whether this holds in vivo and applies to deep hypothermia remains to be tested. Nonetheless, the same study suggested that cooling- induced UPR^ER may be neuroprotective, potentially helping understand the cellular mechanisms underlying the therapeutic benefits of hypothermia. In the clinic, organ cooling is used in transplantation, and therapeutic hypothermia is applied, among others, in stroke or trauma, helping preserve the functions of key organs like the brain or heart (Kutcher et al., 2016; Yenari and Han, 2012). Therefore, understanding how cold elicits UPR^ER may help in discovering interventions mimicking the beneficial effects of cold, but at a thermoneutral temperature. Such preconditioning could be administered to individuals at risk of accidental hypothermia or patients undergoing therapeutic hypothermia, aimed at minimizing potential injuries and enhancing recovery.

Methods

C. elegans strains and maintenance

Unless stated otherwise, animals were maintained as previously described, grown at 20°C on 2% Nematode Growth Media (NGM) agar plates, seeded with the E. coli OP50 bacteria. All strains used in this study are listed in Supplementary file 5A. Synchronized animals were obtained by extracting embryos from gravid adults with a bleaching solution (30% (v/v) sodium hypochlorite (5% chlorine) reagent (ThermoFisher Scientific; 419550010), 750 mM KOH). The embryos were left to hatch in the absence of food into arrested L1 larvae by overnight incubation at room temperature in M9 buffer (42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4).

Cold adaptation

Synchronized L1 larvae were grown at 20°C until reaching the 1-day-old adult stage. Animals were adapted to the cold for 2 hours at 10°C before being transferred to 4°C.

Polysome and ribosome profiling

Polysome profiling and ribosome profiling were performed as previously described (Arnold et al., 2014; Scheckel et al., 2012).

Computational processing of RNA-seq and ribosome profiling

Both RNA-seq and ribosome profiling were processed with the ribo-seq pipeline in ORFik (v1.22.2). The pipeline was run with default parameters and wraps several steps: stripping adapters using fastp (v0.23.4) using the adapter sequence “TGGAATTCTCGGGTGCCAAGG”, mapping reads with STAR (v2.7.11b) and expression (FPKM) estimates using DESeq2(v1.42.1). The code for this pipeline can be found at https://www.bioconductor.org/packages/release/bioc/vignettes/ORFik/inst/doc/Ribo-seq_pipeline.html.

Western blot analysis

Animals were harvested from plates, washed thrice in M9 buffer and pelleted before being snap-frozen in liquid nitrogen. Protein extracts were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in Lysis Buffer (50 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, 5% glycerol (w/vol), 1 mM PMSF, 7 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001)). Debris was removed by centrifugation at 16,100 x g for 20 minutes at 4°C. Protein concentrations were measured by Bradford Assay (Bio-Rad). The required amount of 4x NuPAGE™ LDS Sample Buffer (Invitrogen, NP0007) and 10x NuPAGE Sample Reducing Agent (Invitrogen, NP0004) was added to the protein samples, followed by an incubation at 70°C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane by wet-transfer. Membranes were washed thrice for 5 minutes with PBS-T, blocked for 1 hour in Intercept (TBS) Blocking Buffer (LI-COR, 927-60001), and incubated overnight at 4°C with primary antibodies diluted in the same blocking buffer. The following primary antibodies were used: 1:10,000 monoclonal mouse anti-puromycin (Merck, MABE343), and 1:5,000 polyclonal rabbit anti-actin (Abcam, ab8227). Detection was carried out with IRDye 680RD- conjugated goat anti-mouse secondary antibody (LI-COR Biosciences, 926-68070) or IRDye 800CW-conjugated goat anti-rabbit secondary antibody (LI-COR Biosciences, 926-32211) and infrared imaging (LI-COR Biosciences, Odyssey CLx).

SUnSET assay

A total of 12,000 synchronized L1 larvae were grown at 20°C and adapted to the cold as described earlier or kept at 20°C. Animals were cultivated for 1 or 3 additional days at 4°C or 20°C. The SUnSET assay was carried out essentially as previously described (Arnold et al., 2014), with modifications to assay protein synthesis at 4°C. In brief, animals were washed twice in S-basal, resuspended in 4 ml S-medium, and transferred to a 50 ml Erlenmeyer. An overnight culture of E. coli OP50 was 10x concentrated in S-medium and 750 µl was added to the animals together with 250 µl of 10 mg/ml puromycin (Millipore Sigma, P8833). The animals were grown for 4 hours at 200 rpm before harvesting. Animals were washed thrice with S-basal, pelleted, and snap frozen in liquid nitrogen. Lysates were prepared as described in the western blot procedures and 40 µg of total protein was loaded per well. The incorporation of puromycin into nascent peptides was measured by normalizing band intensities from anti- puromycin to anti-actin antibodies.

Construction of the lips-11::GFP reporter strain

The Plips-11::GFP::unc-54 3’UTR construct was generated via the MultiSite Gateway Technology (Thermo Fisher Scientific, 12537-023). The lips-11 promoter (1140 bp) and GFP (867 bp) were amplified from C. elegans genomic DNA and a plasmid carrying GFP::H2B (pCM1.35) (Merritt et al., 2008), before being inserted into the entry vectors pDONRP4P1R and pDONR221, respectively (oligos are listed in Supplementary file 5B). The resulting entry vectors were recombined along with the entry vector pCM5.37, carrying the unc-54 3’UTR (699 bp) (Merritt et al., 2008), and the destination vector pCFJ150 (Frokjaer-Jensen et al., 2008), carrying chromosome II integration sites together with the unc-119 (+) gene, resulting in the expression clone Plips-11::GFP::unc-54 3’UTR. Transgenic animals were obtained via single-copy integration into the ttTi5605 locus on chromosome II by injecting EG4322 (ttTi5605 II; unc-119(ed3) III) animals with the expression clone (Frokjaer-Jensen et al., 2008).

Microscopy

Animals were grown and adapted to the cold as described or kept at 20°C. Reporter strains were imaged after an additional 1 or 3 days at 20°C or 4°C. Animals were anesthetized in a drop of 5 mM levamisole in M9 buffer on a 2% (w/v) agarose pad, clustered, covered with a cover slip and immediately imaged with the Zeiss AxioImager Z1 microscope. Micrographs were acquired with an Axiocam MRm REV2 CCD camera using the Zen software (Zeiss) and processed with Image J. The specific area that was analyzed for fluorescent intensities, as well as the number of measurements, is indicated in each figure legend.

Induction of ER stress by tunicamycin treatment

Animals were grown at 20°C on NGM agar plates seeded with E. coli OP50. At the 2 day-old adult stage, animals were shifted to plates containing 20 µg/ml tunicamycin (Millipore Sigma, T7765), prepared from a 1 mg/ml stock solution dissolved in DMSO. Animals were grown for 6 hours at 20°C in the presence of tunicamycin. Control animals were treated similarly on plates containing the same volume of DMSO.

Generation of iOP50 RNAi bacteria

Plasmids targeting ire-1, xbp-1, pek-1, or atf-6 were extracted from overnight cultures of E. coli HT115 bacteria derived from the Ahringer library using the QIAprep Spin Miniprep Kit (QIAGEN, 27104) (Kamath et al., 2003). E. coli OP50 bacteria were rendered RNAi competent and chemically competent as previously described (Neve et al., 2020). The resulting iOP50 bacteria were transfected with 1 µl of the purified plasmids derived from the Ahringer library for downstream gene-specific RNAi or with the plasmid vector L4440 for mock RNAi (Fire et al., 1998; Kamath et al., 2003).

RNAi

Gene-specific knockdown was achieved by feeding the animals with bacteria carrying plasmids expressing double-stranded RNA, sourced from either the Vidal or Ahringer libraries (Kamath et al., 2003; Rual et al., 2004). Overnight cultures of E. coli HT115 (for RNAi at 20°C) or E. coli iOP50 bacteria (for RNAi at 4°C) were induced for 1 hour with 1 mM IPTG and seeded on NGM agar plates containing 1 mM IPTG and 50 µg/ml carbenicillin. Synchronized L1 larvae were grown on the RNAi-inducing agar plates at 20°C until reaching the 1 day-old adult stage and were either adapted to the cold as described and kept for 1 or 3 days at 4°C or kept at 20°C for the same duration. Bacteria containing the ‘empty’ L4440 vector were utilized as a mock RNAi control for all experiments.

Dietary supplementation

For choline supplementation assays, a working stock of 200 mg/ml choline (Millipore Sigma, C7527) in water was added to autoclaved NGM media at 55°C to a final concentration of 50 mM. Fatty acid supplemented plates were prepared as previously described with adaptations (Deline et al., 2013). In brief, working stocks of 100 mM were prepared by dissolving palmatic acid (Millipore Sigma, P9767), oleic acid (Millipore Sigma, O7501), and linoleic acid (Millipore Sigma, L8134) in 50% ethanol. Fatty acid sodium salts were added to autoclaved NGM medium containing 0.1% Tergitol (NP40) to a final concentration of 0.8 mM. Fatty acid supplemented plates were covered with foil to prevent light oxidation. All plates were seeded with an overnight culture of E. coli OP50 and dried for 3 days before use.

The assay for C. elegans cold survival

Cold survival experiments were performed as previously described (Pekec et al., 2022). In brief, a minimum of 150 synchronized L1 larvae were grown and adapted to the cold as previously described for each time point. Animals were sampled at the indicated time points and their survival was scored after recovery for 24 hours at 20°C, animals that were unresponsive to touch were considered dead. Cold survival was assessed from six independent biological replicates, with a minimum of 850 animals used to assess the viability at each indicated time point.

RT-qPCR

Approximately 6,000 animals that were adapted to the cold and kept for 1 or 3 days at 4°C or kept at 20°C for the same time were subjected to RNA extraction as previously described (Arnold et al., 2014). Subsequently, genomic DNA was removed by DNase treatment and the quality of the RNA was assessed using the NanoDrop Spectrophotometer. Reverse transcription was performed by using the SuperScript^TM IV First – Strand Synthesis System with random primers, following the protocol from the suppliers. RT-qPCR was performed with 2.5 µl of 1:5 diluted cDNA, 0.25 µl of 10 µM gene-specific primers (Supplementary file 2, Table 3) and 2 µl of the HOT FIREPol EvaGreen qPCR Mix (Solis BioDyne, 08-36-00001) in a LightCycler 96 qPCR machine.

Data availability

RNA sequencing information from this research can be found in the GEO repository under the following accession numbers: GSE269587 (RNA-seq; use the reviewer token mdkdwkiqhfeznkn) and GSE269589 (Ribo-seq; use the reviewer token gnqxucakhryhncd).

Supporting information

Supplementary file 1. Total RNA-seq data of wild-type animals kept at the different indicated temperatures.

Supplementary file 2. Ribo-seq data of wild-type animals kept at the different indicated temperatures.

Supplementary file 3. List of genes highlighted in Figure 2A in red.

Supplementary file 4. IRE-1 responsive genes from published datasets used to generate the Venn diagram in Figure 7 – figure supplement 1. The IRE-1 dependent cold genes were selected from Table S2 and Table S3 from Dudkevich and colleagues (Dudkevich et al., 2022), genes were selected if they were in an upregulated cluster in wild-type animals at 2°C and absent from upregulated clusters in ire-1(ok799) mutants.

Supplementary file 5. Supplemental tables describing the C. elegans strains (5A) and oligos (5B) used in this study.

Acknowledgements

We thank Dimos Gaidatzis and the Functional Genomics and Computational Biology facilities at the Friedrich Miescher Institute for Biomedical Research for the initial genomic analysis. We also thank Agnieszka Chabowska-Kita and the Laboratory of Animal Model Organisms (Institute of Bioorganic Chemistry PAS) for constructing the lips-11 reporter strain.

Additional information

Funding

The research leading to these results received funding from the Norwegian Financial Mechanism 2014–2021 operated by the Polish National Science Center under the project contract nr UMO-2019/34/H/NZ3/00691. Some of the strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Author contributions

Melanie L. Engelfriet: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft.

Yanwu Guo: Conceptualization, Formal analysis, Investigation, Methodology. Andreas Arnold: Investigation, Formal analysis, Methodology, Validation.

Eivind Valen: Formal analysis, Visualization.

Rafal Ciosk: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Visualization, Resources, Writing – original draft.

Declaration of interests

The authors declare no competing interests.