Reprograming gene expression in hibernating C. elegans involves the IRE-1/XBP-1 pathway
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eLife Assessment
This study uses C. elegans, a poikilothermic ("cold-blooded") animal, to investigate the interesting question of how cells and organisms adapt to prolonged exposure to cold temperature. The study employed ribosome profiling and RNAseq analyses and provides a useful inventory of genes changed in cold adapted nematodes. However, the overall conclusions that 1) translation is ongoing at a low rate and 2) IRE mediated transcriptional changes play a significant role in cold adaptation are incompletely supported by the evidence provided. The authors are encouraged to conduct additional bioinformatic analyses and rewrite the manuscript to more accurately reflect the evidence provided.
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Abstract
In the wild, many animals respond to cold temperatures by entering hibernation. In the clinic, controlled cooling is used in transplantation and emergency medicine. Yet, the molecular mechanisms that the cells use to survive cold remain largely unexplored. One aspect of cold adaptation is a global downregulation of protein synthesis. Studying it in the nematode Caenorhabditis elegans , we find that the translation of most mRNAs continues in the cold, albeit at a slower rate, and propose that cold-specific gene expression is regulated primarily at the transcription level. Moreover, we show that the transcription of some cold-induced genes reflects the activation of unfolded protein response (UPR) mediated by the conserved IRE-1/XBP-1 signaling pathway. Our results suggest that the activation of this pathway stems from cold-induced perturbations in proteins and lipids in the endoplasmic reticulum and that its activation is beneficial for cold survival.
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eLife Assessment
This study uses C. elegans, a poikilothermic ("cold-blooded") animal, to investigate the interesting question of how cells and organisms adapt to prolonged exposure to cold temperature. The study employed ribosome profiling and RNAseq analyses and provides a useful inventory of genes changed in cold adapted nematodes. However, the overall conclusions that 1) translation is ongoing at a low rate and 2) IRE mediated transcriptional changes play a significant role in cold adaptation are incompletely supported by the evidence provided. The authors are encouraged to conduct additional bioinformatic analyses and rewrite the manuscript to more accurately reflect the evidence provided.
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Reviewer #1 (Public review):
The manuscript by Engelfriet et.al. addresses an interesting question in animal physiology - how do animals adapt to cold. Using polysome profiling and puromycin labeling, the authors confirm that in C. elegans exposed to a cooling regimen, protein synthesis is decreased globally. They then use RNAseq and ribosome profiling to propose that this decrease is driven mainly by decreased transcription, while translation of most mRNAs continues in the cold at a slower rate. They also find many transcripts whose expression is increased in the cold, and suggest that transcription of some of the cold-induced genes reflects activation of the IRE-1/XBP-1 UPR pathway. The authors further suggest that activation of the UPR by cold is due to cold-induced protein misfolding and perturbations in lipids in the ER, and that …
Reviewer #1 (Public review):
The manuscript by Engelfriet et.al. addresses an interesting question in animal physiology - how do animals adapt to cold. Using polysome profiling and puromycin labeling, the authors confirm that in C. elegans exposed to a cooling regimen, protein synthesis is decreased globally. They then use RNAseq and ribosome profiling to propose that this decrease is driven mainly by decreased transcription, while translation of most mRNAs continues in the cold at a slower rate. They also find many transcripts whose expression is increased in the cold, and suggest that transcription of some of the cold-induced genes reflects activation of the IRE-1/XBP-1 UPR pathway. The authors further suggest that activation of the UPR by cold is due to cold-induced protein misfolding and perturbations in lipids in the ER, and that UPR activation is beneficial for cold survival.
The finding that a decrease in protein synthesis that is characteristic of cold exposure and hibernation is driven primarily by changes in transcription rather than translation is quite interesting and different from findings in other studies. It would be important to understand the reason for this difference. The findings that some of the cold-induced transcription in worms reflects XBP-1-dependent activity of IRE-1 is also new, while UPR activation by lipid perturbations both agrees with previous observations but also exposes differences. The differences highlight the need for better understanding of how different temperature exposures affect different lipids, as cold adaptation is widespread in nature, and cooling is often used in the clinical settings.
However, some concerns with interpretations and technical issues make several major conclusions in this manuscript less rigorous, as explained in detail in comments below. In particular, the two major concerns I have: 1) the contradiction between the strong reduction of global translation, with puromycin incorporation gel showing no detectable protein synthesis in cold, and an apparently large fraction of transcripts whose abundance and translation in Fig. 2A are both strongly increased. 2) The fact that no transcripts were examined for dependance on IRE-1/XBP-1 for their induction by cold, except for one transcriptional reporter, and some weaknesses (see below) in data showing activation of IRE-1/XBP-1 pathway. The conclusion for induction of UPR by cold via specific activation of IRE-1/XBP-1 pathway, in my opinion, requires additional experiments.
Major concerns:
(1) Fig. 1B shows polysomes still present on day 1 of 4{degree sign}C exposure, but the gel in Fig. 1C suggests a complete lack of protein synthesis. Why? What is then the evidence that ribosomal footprints used in much of the paper as evidence of ongoing active translation are from actual translating rather than still bound to transcripts but stationary ribosomes, considering that cooling to 4{degree sign}C is often used to 'freeze' protein complexes and prevent separation of their subunits? The authors should explain whether ribosome profiling as a measure of active translation has been evaluated specifically at 4{degree sign}C, or test this experimentally. They should also provide some evidence (like Western blots) of increases in protein levels for at least some of the strongly cold-upregulated transcripts, like lips-11.
As puromycin incorporation seems to be the one direct measure of global protein synthesis here, it conflicts with much of the translation data, especially considering that quite a large fraction of transcripts have increased both mRNA levels and ribosome footprints, and thus presumably increased translation at 4{degree sign}C, in Fig. 2A.
Also, it is not clear how quantitation in Fig. 1C relates to the gel shown, the quantitation seems to indicate about 50-60% reduction of the signal, while the gel shows no discernable signal.
(2) It is striking that plips-11::GFP reporter is induced in day 1 of 4{degree sign}C exposure, apparently to the extent that is similar to its induction by a large dose of tunicamycin (Fig. 3 supplement), but the three IRE-1 dependent UPR transcripts from Shen 2005 list were not induced at all on day 1(Fig. 4 supplement). Moreover, the accumulation of the misfolded CPL-1 reporter, that was interpreted as evidence that misfolding may be triggering UPR at 4{degree sign}C, was only observed on day 1, when the induction of the three IRE-1 targets is absent, but not on day 3, when it is stronger. How does this agree with the conclusion of UPR activation by cold via IRE-1/XBP-1 pathway? It is true that the authors do note very little overlap between IRE-1/XBP-1-dependent genes induced by different stress conditions, but for most of this paper, they draw parallels between tunicamycin-induced and cold-induced IRE-1/XBP-1 activation.
The conclusion that "the transcription of some cold-induced genes reflects the activation of unfolded protein response (UPR)..." is based on analysis of only one gene, lips-11. No other genes were examined for IRE-1 dependence of their induction by cold, neither the other 8 genes that are common between the cold-induced genes here and the ER stress/IRE-1-induced in Shen 2005 (Venn diagram in Figure 7 supplement), nor the hsp-4 reporter. What is the evidence that lips-11 is not the only gene whose induction by cold in this paper's dataset depends on IRE-1? This is a major weakness and needs to be addressed.
Furthermore, whether induction by cold of lips-11 itself is due to IRE1 activation was not tested, only a partial decrease of reporter fluorescence by ire-1 RNAi is shown. A quantitative measure of the change of lips-11 transcript in ire-1 and xbp-1 mutants is needed to establish if it depends on IRE-1/XBP-1 pathway.
The authors could provide more information and the additional data for the transcripts upregulated by both ER stress and cold, including the endogenous lips-11 and hsp-4 transcripts: their identity, fold induction by both cold and ER stress, how their induction is ranked in the corresponding datasets (all of these are from existing data), and do they depend on IRE-1/XBP-1 for induction by cold? Without these additional data, and considering that the authors did not directly measure the splicing of xbp-1 transcript (see comment for Fig. 3 below), the conclusion that cold induces UPR by specific activation of IRE-1/XBP-1 pathway is premature.
There are also technical issues that are making it difficult to interpret some of the results, and missing controls that decrease the rigor of conclusions:
(1) For RNAseq and ribosome occupancy, were the 20{degree sign}C day 1 adult animals collected at the same time as the other set was moved to 4{degree sign}C, or were they additionally grown at 20{degree sign}C for the same length of time as the 4{degree sign}C incubations, which would make them day 2 adults or older at the time of analysis? This information is only given for SUnSET: "animals were cultivated for 1 or 3 additional days at 4{degree sign}C or 20{degree sign}C". This could be a major concern in interpreting translation data: First, the inducibility of both UPR and HSR in worms is lost at exactly this transition, from day 1 to day 2 or 3 adults, depending on the reporting lab (for example Taylor and Dillin 2013, Labbadia and Morimoto, 2015, De-Souza et al 2022). How do authors account for this? Would results with reporter induction, or induction of IRE-1 target genes in Fig. 4, change if day 1 adults were used for 20{degree sign}C?
Second, if animals at the time of shift to 4{degree sign}C were only beginning their reproduction, they will presumably not develop further during hibernation, while an additional day at 20{degree sign}C will bring them to the full reproductive capacity. Did 4{degree sign}C and 20{degree sign}C animals used for RNAseq and ribosome occupancy have similar numbers of embryos, and were the embryos at similar stages? If embryos were retained in one condition vs the other, how much would they contribute in terms of transcripts, and do the authors expect the same adaptive programs operating in embryos and in the adults?
(2) Second, no population density is given for most of the experiments, despite the known strong effects of crowding (high pheromone) on C. elegans growth. From the only two specifics that are given, it seems that very different population sizes were used: for example, 150 L1s were used in survival assay, while 12,000 L1s in SUnSET. Have the authors compared results they got at high population densities with what would happen when animals are grown in uncrowded plates? At least a baseline comparison in the beginning should have been done.
(3) Fig. 3: it is unclear why the accepted and well characterized quantitative measure of IRE1 activation, the splicing of xbp-1transcript, is not determined directly by RT-PCR. The fluorescent XBP-1spliced reporter, to my knowledge, has not been tested for its quantitative nature and thus its use here is insufficient.
Furthermore, the image of this fluorescent reporter in Fig. 3b shows only one anterior-most row of cells of intestine, and quantitation was done with 2 to 5 nuclei per animal, while lips-11 is induced in entire intestine. Was there spliced XBP-1 in the rest of the intestinal nuclei? Could the authors show/quantify the entire animal (20 intestinal cells) rather than one or two rows of cells?
(4) The differences in the outcomes from this study and the previous one (Dudkevich 2022) that used 15{degree sign}C to 2{degree sign}C cooling approach are puzzling, as they would suggest two quite different IRE-1 dependent programs of cold tolerance. It would be good if authors commented on overlapping/non-overlapping genes, and provided their thoughts on the origin of these differences considering the small difference in temperatures. Second, have the authors performed a control where they reproduced the rescue by FA supplementation of poor survival of ire-1 mutants after the 15{degree sign}C to 2{degree sign}C shift?
Without this or another positive control, and without measuring change in lipid composition in their own experiments, it is unclear whether the different outcomes with respect to FAs are due to a real difference in adaptive programs at these temperatures, or to failure in supplementation?
(5) Have the authors tested whether and by how much ire-1(ok799) mutation shortens the lifespan at 20{degree sign}C? This needs to be done before the defect in survival of ire-1 mutants in Fig. 7a can be interpreted.
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Reviewer #2 (Public review):
Summary:
This study investigates cold induced states in C. elegans, using polysome profiling and RNA seq to identify genes that are differentially regulated and concluding that cold-specific gene regulation occurs at the transcriptional level. This study also includes analysis of one gene from the differentially regulated set, lips-11 (a lipase), and finds that it is regulated in response to a specific set of ER stress factors.
Strengths:
(1) Understanding how environmental conditions are linked to stress pathways is generally interesting.
(2) The study used well-established genetic tools to analyze ER stress pathways.
Weaknesses:
(1) The conclusions regarding a general transcriptional response are based on one gene, lips-11, which does not affect survival in response to cold. We would suggest altering the …
Reviewer #2 (Public review):
Summary:
This study investigates cold induced states in C. elegans, using polysome profiling and RNA seq to identify genes that are differentially regulated and concluding that cold-specific gene regulation occurs at the transcriptional level. This study also includes analysis of one gene from the differentially regulated set, lips-11 (a lipase), and finds that it is regulated in response to a specific set of ER stress factors.
Strengths:
(1) Understanding how environmental conditions are linked to stress pathways is generally interesting.
(2) The study used well-established genetic tools to analyze ER stress pathways.
Weaknesses:
(1) The conclusions regarding a general transcriptional response are based on one gene, lips-11, which does not affect survival in response to cold. We would suggest altering the title, to replace "Reprograming gene expression: with" Regulation of the lipase lips-11".
(2) There is no gene ontology with the gene expression data.
(3) Definitive conclusions regarding transcription vs translational effects would require use of blockers such as alpha amanatin or cyclohexamide.
(4) Conclusions regarding the role of lipids are based on supplementation with oleic acid or choline, yet there is no lipid analysis of the cold animals, or after lips-1 knockdown. Although choline is important for PC production, adding choline in normal PC could have many other metabolic impacts and doesn't necessarily implicate PC with out lipidomic or genetic evidence.
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Reviewer #3 (Public review):
Summary:
The authors sought to understand the molecular mechanisms that cells use to survive cold temperatures by studying gene expression regulation in response to cold in C. elegans. They determined whether gene expression changes during cold adaptation occur primarily at the transcriptional level and identified specific pathways, such as the unfolded protein response pathway, that are activated to possibly promote survival under cold conditions.
Strengths:
Effective use of bulk RNA sequencing (RNA-seq) to measure transcript abundance and ribosome profiling (ribo-seq) to assess translation rates, providing a comprehensive view of gene expression regulation during cold adaptation. This combined approach allows for correlation between mRNA levels and their translation, thereby offering evidence for the …
Reviewer #3 (Public review):
Summary:
The authors sought to understand the molecular mechanisms that cells use to survive cold temperatures by studying gene expression regulation in response to cold in C. elegans. They determined whether gene expression changes during cold adaptation occur primarily at the transcriptional level and identified specific pathways, such as the unfolded protein response pathway, that are activated to possibly promote survival under cold conditions.
Strengths:
Effective use of bulk RNA sequencing (RNA-seq) to measure transcript abundance and ribosome profiling (ribo-seq) to assess translation rates, providing a comprehensive view of gene expression regulation during cold adaptation. This combined approach allows for correlation between mRNA levels and their translation, thereby offering evidence for the authors' conclusion that transcriptional regulation is the primary mechanism of cold-specific gene expression changes.
Weaknesses:
The study has several weaknesses: it provides limited novel insights into pathways mediating transcriptional regulation of cold-inducible genes, as IRE-1 and XBP-1 are already well-known responders to endoplasmic reticulum stress, including that induced by cold. Additionally, the weak cold sensitivity phenotype observed in ire-1 mutants casts doubt on the pathway's key role in cold adaptation. The study also overlooks previous research (e.g. PMID: 27540856) that links IRE-1 to SKN-1, another major stress-responsive pathway, potentially missing important interactions and mechanisms involved in cold adaptation.
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