Drosophila cap-binding protein eiF4EHP promotes translation via a 3’UTR-dependent mechanism under hypoxia and contributes to fruit fly adaptation to oxygen variations
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Abstract
Hypoxia induces profound modifications in gene expression program enabling eukaryotic cells to adapt to lowered ATP supply resulting from the blockade of oxidative phosphorylation. One major consequence of oxygen deprivation is the massive repression of protein synthesis, leaving a limited set of mRNAs to be translated. D. melanogaster is strongly resistant to oxygen fluctuations, however the mechanisms allowing specific mRNA to be translated in hypoxia are still unknown. Here, we show that Ldh mRNA encoding lactate dehydrogenase is highly translated in hypoxia by a mechanism involving its 3’ untranslated region. Furthermore, we identified the cap-binding protein eiF4HP as a main factor involved in 3’UTR-dependent translation under hypoxia. In accordance with this observation, we show that eiF4EHP is necessary for Drosophila development under low oxygen concentrations and contributes to Drosophila mobility after hypoxic challenge. Altogether, our data bring new insight into mechanisms contributing to Drosophila adaptation to oxygen variations.
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Reply to the reviewers
1. Description of the planned revisions
Suggested by reviewer 3:
- The authors showed the importance of the 3'UTR of Ldh in regulating Ldh translation under hypoxia, and they mention in the discussion that further investigation is needed to understand the nature of this regulation. Mutational analysis of the 3'UTR sequence could help to extend the paper and enhance its impact. *
*A mutational analysis of Ldh mRNA 3’UTR is currently under way and will be included in the revised version of the manuscript. *
2.One obvious shortcoming of this paper is that the functions of the Ldh 3' UTR and or eIF4EHP are* not connected by experimental tests. …
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Reply to the reviewers
1. Description of the planned revisions
Suggested by reviewer 3:
- The authors showed the importance of the 3'UTR of Ldh in regulating Ldh translation under hypoxia, and they mention in the discussion that further investigation is needed to understand the nature of this regulation. Mutational analysis of the 3'UTR sequence could help to extend the paper and enhance its impact. *
*A mutational analysis of Ldh mRNA 3’UTR is currently under way and will be included in the revised version of the manuscript. *
2.One obvious shortcoming of this paper is that the functions of the Ldh 3' UTR and or eIF4EHP are* not connected by experimental tests. Experiments aimed at determining the functional relation of the 3' UTR and eIF4EHP could enhance the paper and deliver a more complete story about the mechanism of selective translation under hypoxia.*
We believe that data presented in figure 4 do functionally connect eIF4EHP and Ldh 3’UTR since we show that a reporter mRNA containing the Ldh mRNA 3’UTR is specifically recruited on polysome in hypoxic control cells but not in eIF4EHP-depleted cells. However, we plan to further strengthen this demonstration by analyzing Ldh mRNA 3’UTR mutants in polysomes of WT or eIF4EHP-deficient cells.
*3.The authors refer to human cell studies showing that HIF2a is involved in mRNA translation in hypoxic conditions. They mentioned in the discussion that Drosophila Sima has not been identified to interact with eIF4EHP. To test whether Sima regulates Ldh translation, the authors could test the involvement of Sima in experiments from Fig 6. *
As suggested, we plan to inhibit Sima expression by CRISPR/Cas9 in S2 cells and if viable, evaluate the recruitment of eiF4EHP on polysomes of hypoxic Sima-KO cells. The interaction of Ldh mRNA with eiF4EHP in absence of Sima could also be tested. However, the later experiment is most probably impossible as the transcriptional induction of Ldh by hypoxia is strongly reduced upon Sima depletion in S2 cells (Dekanty A et al 2010, Plos Genet 6(6) e1000994) and Drosophila larvae (Wang et al. 2016, ELife:e18126).
2.Description of the revisions that have already been incorporated in the transferred manuscript* *
Reviewer 1
*5.The authors should integrate the emerging concept of adaptive cap-binding translation factors in the discussion. It is still generally assumed that inhibition of eIF4E results automatically to cap-independent protein synthesis. The discovery that eIF3D, 4E2 and 4E3, amongst others, can initiate stimuli-specific translation should be discussed in the context of the work from this paper. *
The discussion now includes the emerging concept of diverse non-canonical cap-dependent translation mechanisms.
Reviewer 2
Minor comments:
- Similarly, in Fig 6C it would be good to show the equivalent CLIP data for 21% oxygen. Presumably, since eIF4HP is not in polysomes in the normoxic condition, there should be no enrichment (or little enrichment) for the Ldh mRNA. *
Fig. 6C has been revised to indicate more clearly that the data were obtained in cells under hypoxia. The method section has been modified to include a reference protocol. A statistical test has been included. Ldh mRNA is expressed at very low level in normoxic S2 cells and is strongly induced upon hypoxic exposure (>500 fold). Therefore, the comparison of eiF4EHP binding to Ldh mRNA between normoxic and hypoxic conditions is not relevant due to this strong difference in Ldh mRNA abundance.
*For Fig 7B - it's a bit confusing to label the y-axis as "flies escaping" to mean flies that climb past a certain limit. I suggest relabeling the axis to something like "flies climbing past threshold". *
The proposed modification has been introduced in Fig.7B.
Reviewer 3
- In Fig 3A-B, induction of LDH by hypoxia is almost completely blocked by eiF4EHP knockdown in fly heads but not in S2 cells. We wonder whether this indicates tissue specific regulation of LDH translation, such that there might be alternative cap-binding proteins in S2 cells. Please comment.*
Expression data from flyRNAi and our RNAseq experiments (de Toeuf et al, Scientific Reports, 2018) indicate that only eiF4E1, eiF4E6 and eiF4EHP are expressed in S2 cells in normoxia and hypoxia, all the other members (eIF4E3, 4, 5, 7) being very weakly or not expressed at all. We show that eiF4E6 KO does not impair Ldh mRNA association to polysomes in S2 under hypoxia (Fig.3E,F), thereby suggesting that this member of the family is not involved in Ldh mRNA hypoxic translation. Therefore, the difference in LDH suppression resulting from eiF4EHP KO in S2 versus fly heads cannot be explained by the involvement of alternative eiF4E family members in S2 cells*. *
- In Fig 3E, the authors present results of puromycin incorporation with eIF4EHP KD under hypoxia. It is actually not clear what the function of eIF4EHP is under normoxic conditions. The authors shall include a control of puromycin incorporation with eIF4EHP KD under 21% O2. In addition, the authors should include statistical tests for the comparisons.*
These data are now presented in fig. 3G. We have increased the number of replicates (now n=12) and, as suggested, we compared the incorporation of puromycin in normoxic and hypoxic condition. We observed a stronger reduction in puromycin incorporation in eIF4EHP KO cells as compared to controls when cells are exposed to hypoxic conditions. Our results therefore suggest that the positive effect of eiF4EHP on protein synthesis is predominant under hypoxic conditions. *
- In Fig 4C, the authors did Western Blots to detect eIF4EHP, and find that there is protein signal under the condition with eIF4EHP KD + D. persimilis eIF4EHP overexpression. It is unclear whether the antibody detects both eIF4EHPs in Drosophila melanogaster and D. persimilis, or whether, alternatively, expressing the D. persimilis version induces cells to express endogenous eIF4EHP. Please comment. * The antibody detects both eiF4EHP, with DpeiF4EHP-V5 being slightly heavier. The difference of band intensity between WT+ DpeiF4EHP and KO + DpeiF4EHP most probably results from a difference of DpeiF4EHP construct transfection efficiency in the two cell lines. This difference of expression has no influence on results shown in Fig.4D,E.
The antibody used in Fig.4C and its cross-reactivity to dpeiF4EHP have been specified in the figure legend.
- In Fig 7A, we don't see the point of including results of flies carrying balancers as control. Direct comparisons with mCherry-RNAi should suffice. Also, presenting the percentage of hatched embryos can be misleading. We would suggest the authors present the absolute numbers of embryos examined and indicate the number of larvae that hatched.*
We believe that our experimental protocol is valid and supports our conclusions. The presence of the balancer chromosome provides an internal control for each cross. *The balancer being transmitted in 50% of the embryos we can directly compare for each individual cross, after eclosion, the number of individuals bearing or not the dominant balancer marker. We have performed an additional experiment to increase the sample size for hypoxic conditions and we now provide a statistical analysis. *
- In addition (Fig 7) "birth" is not an appropriate term for insects. Please state whether the numbers indicated larvae "hatched" from eggs, or adult flies "eclosed" from pupae. Please use these terms in the text, figure and figure legends. *
These terms have been replaced in the revised manuscript.
- In Fig 6C, a statistical test is missing. *
A statistical test has been included*. *
- The authors sometimes refer to knockdown as KO, please be accurate.*
This point has been revised. We define KO upon gene disruption by CRISPR/cas9 as performed in S2 cells and KD upon Knock down by RNAi as performed in flies.
Reviewer 4
- In human cells eIF4E2 (the eIF4EHP orthologue) can activate translation in hypoxia by forming a complex with HIF2alpha, RBM4, and eIF4G3. The paper states in the discussion that inhibition of the Drosophila eIF4G3 counterparts eIF4G2 and NAT1 does not affect translation under hypoxia. However, the data behind this important conclusion are not shown, nor are details given as to how eIF4G2 and NAT1 were 'inhibited', whether or not both were inhibited at the same time, and whether the fly RBM4 orthologue Lark was investigated. While the mechanism of how eIF4EHP activates translation must be different in Drosophila from that in human cells because of the absence of HIF2alpha, not much more can be concluded than that in the absence of explicit experimental data. *
We tested the role of eIF4G2 and NAT1 by CRISPR/cas9 inactivation independently in S2 cells under hypoxia. We did not observe any modification in LDH synthesis and Ldh mRNA distribution in polysomes. These experiments are mentioned in the discussion. We tested Lark KO in S2 and Lark KD in flies. We observed in both settings that Lark inactivation is lethal, thereby precluding the study of Lark in hypoxic translation by gene inactivation*. *
- Fig 3B shows a large increase in LDH levels under hypoxic conditions in eIF4EHP KO cells, which is inconsistent with the narrative description of this result and the paper's conclusions. Ratios of LDH in normoxic and hypoxic conditions for eIF4EHP KO and control cells should be quantitated from multiple experiments, compared, and tested for statistical significance. * *The results now shown in Fig.3C,D are crucial and are consistent with the main conclusion of the paper that eiF4EHP activates Ldh mRNA translation in a 3’UTR-dependent manner. *
*Ratios of LDH/Actin levels have now been measured in 7 independent experiments and tested for statistical significance. We observed a significant decrease of hypoxia induced LDH production in eIFEHP KO cell line as compared to Cas9 control cell line. *
- Statistical significance should also be shown for the data in Fig 3E. I note that the empty vector control reduces puromycin incorporation by quite a lot. *
*The experiment (now presented in fig. 3G) has been reproduced (from n=3 to n=6). A statistical analysis has been performed. The cell line transfected with the empty vector is the adequate control and reduction of puromycin incorporation in these cells in comparison to untransfected cells might results from cas9 expression. The figure was modified to present comparisons between KO and control cell lines both in hypoxic and normoxic conditions. *
- The number of survivors is very low for both control and eIF4EHP KD flies in Fig 7A. Are these comparisons statistically significant? *
This experiment was repeated to increase the number of flies in the hypoxic group. Statistical significance is now indicated and the legend was modified accordingly.
- It should be explained why eIF4E6 KO was included in Fig 3. *
This explanation is now included in the text.
- I assume that 6% oxygen was used for the viability experiment in Fig 7A because the lower concentration of 1% used in all other experiments would be lethal to both control and KD flies. This reasoning should be made explicit. *
This is in fact the reason for using 6% oxygen. It is indicated in the revised manuscript.
*3. There is a great deal of sloppiness about nomenclature. The FlyBase term for the molecule of interest is eIF4EHP. Within this manuscript I found it referred to as eIF4EHP, eiF4EHP, eIF4HP, eiF4HP, and ei4EHP. This requires correction. * The nomenclature has been revised according to FlyBase terms.
- The references are also sloppy and presented in several different formats*.
The reference list has been revised and is now homogenously formatted.
2. Description of analyses that authors prefer not to carry out
Experiments suggested by Reviewer 1:
*1.The work focuses mainly on LDH. It would be a missed opportunity to show the effect of eIF4EHP in hypoxic Drosophila on puromycin incorporation. *
This experiment has been performed. However, puromycin incorporation is only detectable in normoxic larvae. Puromycin labeling is undetectable in hypoxic larvae or adult flies. This is most likely due to 2 distinct parameters that make the experiment irrelevant. First, translational activity is inhibited in hypoxia (the measured parameter) and second, the feeding activity of larvae and adults is also strongly reduced or totally stopped in hypoxia, therefore drastically reducing the uptake of puromycin compared to normoxic individuals and making the comparison of the 2 conditions (normoxia vs hypoxia) impossible.
*We decided not to further pursue this approach. *
*2.In mammalian cells, blocking de novo transcription does not affect protein accumulation of eIF4E2 translated mRNA, even if these mRNA do not increase during hypoxia. It would be important to silence sima and test if this could block increased translation of ldh or other target in hypoxic conditions. This would suggest that sima is both a transcription and translation factor that evolved in HIF1a and HIF2a, the latter being a hypoxic translation regulator. This can be compared to cells treated with a general transcription inhibitor. These experiments would broaden the impact of the work. *
*Here, reviewer 1 is most certainly referring to figure 1 of Uniacke et al. 2012 (Nature 486:126) where the authors observed that human EGFR protein accumulates in hypoxic human U87MG cells treated or not with actinomycin D in a HIF2-dependent manner. Supplementary Fig.3 from the same paper clearly shows that EGFR mRNA is constitutively present in U87MG independently of hypoxic exposure or actinomycin D treatment. The same type of experiment cannot be transposed in for LDH synthesis in Drosophila cells. Indeed, Ldh mRNA is strongly induced at the transcriptional level upon hypoxia. Therefore, inhibiting Sima expression or treating cells will Actinomycin D will block Ldh mRNA synthesis making it impossible to analyze further its translational status. *
*3.Fig 5C. It is unclear what are the conclusions of the authors on the lack of co-localization between poly(A)RNA and eIF4EHP. In principle, eIF4EHP should co-localize with poly(A) mRNA. Perhaps a proximity ligation assay should be done to clarify this question. The data shown in F5C is not convincing one way or the other and needs clear cut experiments. Idem for 5A and B. PLA would provide convincing results. *
*We observed a reduced puromycin incorporation in eIF4EHP KO cells under hypoxic conditions (Fig. 3E), revealing a decrease in protein synthesis activity in hypoxic cells depleted of eiF4EHP. Therefore, our observations support a model in which eiF4HP is promoting translation of specific mRNA targets under hypoxic conditions rather than acting as a global translational inhibitor. In this context, it is expected, as confirmed in figure 5C, that eiF4EHP will not colocalize with poly(A)+ mRNA in hypoxic S2 cells as most of these mRNA are not translated under these conditions (Fig1 A,B). The paragraph describing fig. 5C has been modified to clearly specify this point. *
- Fig 6C is important but the data is hard to understand. First, the Y-axis is "enrichment relative to input". Is this hypoxia vs normoxia? Is the Y-axis log (probably)? The best would be to show that normoxia and hypoxia and see if eIF4EHP binds to ldh mRNA in both conditions perhaps acting as a translational repressor in normoxia and translational activator in hypoxia. Also, the authors could pool monosome and polysome fractions and see in which fractions eIF4EHP binds to Ldh mRNA. Finally, do the authors think that rpl32 remains associated with ldh mRNA in normoxia and hypoxia? *
*Fig.6C (linear Y-axis) shows that Ldh mRNA is specifically bound by eiF4EHP in S2 cells under hypoxia as it is immunoprecipitated with eiF4EHP in WT cells and not in eIF4EHP KO S2. The Ldh mRNA/eiF4EHP interaction is specific as rpl32 mRNA is not immunoprecipitated in the same conditions. *
*Ldh mRNA is barely expressed in normoxia, rendering the analysis of Ldh mRNA binding to eiF4EHP technically difficult and introducing a strong bias to the relative comparison of this association in hypoxia versus normoxia. *
Our data showing that Ldh mRNA is massively associated to polysomes under hypoxia (fig.1D,E) and that this association is strongly impaired in eiF4EHP KO cells (Fig. 3C,D) combined to Ldh mRNA direct association to eiF4EHP in hypoxia (Fig.6C) strongly support the role of eiF4EHP in Ldh mRNA translation. Association of Ldh mRNA to eiF4EHP in polysomes versus monosomes would not significantly contribute to our conclusions.
Experiments suggested by Reviewer 2:
Minor comments:
- Fig 2A - it would be good to show the equivalent luciferase assays at 21% oxygen, to test whether the elevated activity of the Ldh 3'UTR is something specific to the hypoxic condition, or whether this is always the case, also under non-stressed conditions.*
*The reporter gene assays of fig2A have been performed with luciferase reporters placed downstream of the Ldh gene promoter whose activity is very low under normoxia. This strategy was used to avoid luciferase expression and accumulation prior to exposure to hypoxia and potential masking of the Ldh 3’UTR effect on luciferase activity. The corresponding paragraphs have been modified to explicitly describe that reporter constructs used in this experiment are hypoxia-inducible. *
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Referee #4
Evidence, reproducibility and clarity
Summary:
The manuscript by Liang et al. shows that, while overall translation is reduced under hypoxic conditions, translation of Ldh mRNA substantially increases. This increase is demonstrated to depend upon the Ldh 3' UTR and the variant translation factor eIF4EHP. The manuscript further shows that eIF4EHP associates with polysomal fractions and with Ldh mRNA under hypoxic conditions and that it is enriched in cytoplasmic foci apparently distinct from stress granules and P bodies in hypoxia. Finally, the paper provides evidence that loss of eIF4EHP worsens the survival rate of flies in reduced oxygen conditions.
Major comments:
T…
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Referee #4
Evidence, reproducibility and clarity
Summary:
The manuscript by Liang et al. shows that, while overall translation is reduced under hypoxic conditions, translation of Ldh mRNA substantially increases. This increase is demonstrated to depend upon the Ldh 3' UTR and the variant translation factor eIF4EHP. The manuscript further shows that eIF4EHP associates with polysomal fractions and with Ldh mRNA under hypoxic conditions and that it is enriched in cytoplasmic foci apparently distinct from stress granules and P bodies in hypoxia. Finally, the paper provides evidence that loss of eIF4EHP worsens the survival rate of flies in reduced oxygen conditions.
Major comments:
The experimental data largely support the conclusions that are drawn but I have some important reservations.
In human cells eIF4E2 (the eIF4EHP orthologue) can activate translation in hypoxia by forming a complex with HIF2alpha, RBM4, and eIF4G3. The paper states in the discussion that inhibition of the Drosophila eIF4G3 counterparts eIF4G2 and NAT1 does not affect translation under hypoxia. However, the data behind this important conclusion are not shown, nor are details given as to how eIF4G2 and NAT1 were 'inhibited', whether or not both were inhibited at the same time, and whether the fly RBM4 orthologue Lark was investigated. While the mechanism of how eIF4EHP activates translation must be different in Drosophila from that in human cells because of the absence of HIF2alpha, not much more can be concluded than that in the absence of explicit experimental data.
Fig 3B shows a large increase in LDH levels under hypoxic conditions in eIF4EHP KO cells, which is inconsistent with the narrative description of this result and the paper's conclusions. Ratios of LDH in normoxic and hypoxic conditions for eIF4EHP KO and control cells should be quantitated from multiple experiments, compared, and tested for statistical significance.
Statistical significance should also be shown for the data in Fig 3E. I note that the empty vector control reduces puromycin incorporation by quite a lot.
The number of survivors is very low for both control and eIF4EHP KD flies in Fig 7A. Are these comparisons statistically significant?
Minor comments:
It should be explained why eIF4E6 KO was included in Fig 3.
I assume that 6% oxygen was used for the viability experiment in Fig 7A because the lower concentration of 1% used in all other experiments would be lethal to both control and KD flies. This reasoning should be made explicit.
There is a great deal of sloppiness about nomenclature. The FlyBase term for the molecule of interest is eIF4EHP. Within this manuscript I found it referred to as eIF4EHP, eiF4EHP, eIF4HP, eiF4HP, and ei4EHP. This requires correction.
The references are also sloppy and presented in several different formats.
Significance
As mentioned above, it is established in human cells that the orthologue of eIF4EHP can activate translation under hypoxic conditions. So the basic result in this manuscript simply confirms that the same phenomenon occurs in flies. As mentioned in the previous section, in human cells eIF4EHP activates translation in a 3' UTR dependent manner as part of a complex containing HIF2alpha, RBM4, and eIF4G3. While a different mechanism is likely to exist in Drosophila, it is not elucidated by the experimental data presented in this paper. Therefore I find the work to be of limited significance.
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Referee #3
Evidence, reproducibility and clarity
Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical …
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Referee #3
Evidence, reproducibility and clarity
Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical component for this hypoxia induced translation, and show that its function is important for fly survival and development under hypoxic conditions. The authors present extensive, compelling results to support their conclusions. However, we still find this work can be improved in several aspects:
The authors showed the importance of the 3'UTR of Ldh in regulating Ldh translation under hypoxia, and they mention in the discussion that further investigation is needed to understand the nature of this regulation. Mutational analysis of the 3'UTR sequence could help to extend the paper and enhance its impact.
One obvious shortcoming of this paper is that the functions of the Ldh 3' UTR and o eIF4EHP are not connected by experimental tests. Experiments aimed at determining the functional relation of the 3' UTR and eIF4EHP could enhance the paper and deliver a more complete story about the mechanism of selective translation under hypoxia.
The authors refer to human cell studies showing that HIF2a is involved in mRNA translation in hypoxic conditions. They mentioned in the discussion that Drosophila Sima has not been identified to interact with eIF4EHP. To test whether Sima regulates to Ldh translation, the authors could test the involvement of Sima in experiments from Fig 6.
In Fig 3A-B, induction of LDH by hypoxia is almost completely blocked by eiF4EHP knockdown in fly heads but not in S2 cells. We wonder whether this indicates tissue specific regulation of LDH translation, such that there might be alternative cap-binding proteins in S2 cells. Please comment.
In Fig 3E, the authors present results of puromycin incorporation with eIF4EHP KD under hypoxia. It is actually not clear what the function of eIF4EHP is under normoxic conditions. The authors shall include a control of puromycin incorporation with eIF4EHP KD under 21% O2. In addition, the authors should include statistical tests for the comparisons.
In Fig 4C, the authors did Western Blots to detect eIF4EHP, and find that there is protein signal under the condition with eIF4EHP KD + D. persimilis eIF4EHP overexpression. It is unclear whether the antibody detects both eIF4EHPs in Drosophila melanogaster and D. persimilis, or whether, alternatively, expressing the D. persimilis version induces cells to express endogenous eIF4EHP. Please comment.
In Fig 7A, we don't see the point of including results of flies carrying balancers as control. Direct comparisons with mCherry-RNAi should suffice. Also, presenting the percentage of hatched embryos can be misleading. We would suggest the authors present the absolute numbers of embryos examined and indicate the number of larvae that hatched.
In addition (Fig 7) "birth" is not an appropriate term for insects. Please state whether the numbers indicated larvae "hatched" from eggs, or adult flies "eclosed" from pupae. Please use these terms in the text, figure and figure legends.
In Fig 6c, a statistical test is missing.
The authors sometimes refer to knockdown as KO, please be accurate.
Significance
Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical component for this hypoxia induced translation, and show that its function is important for fly survival and development under hypoxic conditions. These results are unique and significant, and should be of interest to researchers who work on hypoxia, stress responses in general, and mRNA translation. As noted above (#1, #2) the paper could go further mechanistically, to determine the relative roles of the LDH 3' UTR and eIF4EHP. But it already has an extensive array of impressive translation data.
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Referee #2
Evidence, reproducibility and clarity
Summary:
When cells are stressed, such as during hypoxia, most of the canonical mRNA translation is shut off. Nonetheless, stress-response genes need to be translated. One such example is the lactate dehydrogenase (Ldh) mRNA, which encodes for an enzyme needed by cells to resist hypoxia. Therefore, these mRNA employ non-canonical mechanisms of translation to escape the general repression. Here, Liang et al. study the molecular mechanism how the Ldh mRNA is translated in Drosophila during hypoxia. They discover that the Ldh 3'UTR contains sequences that enable efficient translation. They identify the non-canonical initiation factor …
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Referee #2
Evidence, reproducibility and clarity
Summary:
When cells are stressed, such as during hypoxia, most of the canonical mRNA translation is shut off. Nonetheless, stress-response genes need to be translated. One such example is the lactate dehydrogenase (Ldh) mRNA, which encodes for an enzyme needed by cells to resist hypoxia. Therefore, these mRNA employ non-canonical mechanisms of translation to escape the general repression. Here, Liang et al. study the molecular mechanism how the Ldh mRNA is translated in Drosophila during hypoxia. They discover that the Ldh 3'UTR contains sequences that enable efficient translation. They identify the non-canonical initiation factor eIF4HP as a factor required for Ldh translation, and show that it binds the Ldh mRNA. Interestingly, they show that the ability of the Ldh 3'UTR to promote translation during hypoxia depends on eIF4HP. They go on to show that unlike other canonical translation initiation factors, during hypoxia eIF4HP remains in polysomes and does not form translationally-repressed aggregates such as stress granules or P-bodies. Finally, Liang et al show that eIF4HP is required in flies to efficiently resist hypoxia.
Major comments:
This is a very nice and solid study. Overall, I found the data and the conclusions very convincing. Also nice is how they cover the topic broadly, starting at the molecular level with the 3'UTR of the Ldh mRNA and ending with physiological assays such as resistance of flies to hypoxia. In particular I found the experiments presented in Fig 2C and 4A to be very nice, showing that the 3'UTR is responsible for the resistance to translation inhibition, and that this is mediated by eiF4EHP. I have only a few minor comments (below) to strengthen further the study, but I think it could be published also without these additional experiments.
Minor comments:
I have only two minor suggestions for experiments that would further strengthen the conclusions presented in the paper:
• Fig 2A - it would be good to show the equivalent luciferase assays at 21% oxygen, to test whether the elevated activity of the Ldh 3'UTR is something specific to the hypoxic condition, or whether this is always the case, also under non-stressed conditions.
• Similarly, in Fig 6C it would be good to show the equivalent CLIP data for 21% oxygen. Presumably, sinc eIF4HP is not in polysomes in the normoxic condition, there should be no enrichment (or little enrichment) for the Ldh mRNA.
• For Fig 7B - it's a bit confusing to label the y-axis as "flies escaping" to mean flies that climb past a certain limit. I suggest relabeling the axis to something like "flies climbing past threshold".
Significance
• The mechanisms of translation that one can read when opening a standard molecular biology textbook are the canonical mechanisms that take place in cells when they are not stressed. Cells, however, often experience stress. For instance, animals in the wild are exposed to heat or cold stress, cancer cells in a tumor are exposed to hypoxia, low amino acids, low sugar, etc. In such conditions, the canonical mRNA translation systems are shut down, and non-canononical mechanisms are remain (or are turned on). So it is a very interesting topic to understand how these non-canonical mRNA translation mechanisms function. This study contributes significantly to this topic by identifying the molecular mechanism how Ldh is translated during hypoxia. Ldh is an important gene because it is the last step of anaerobic glycolysis in animals, needed for cells to produce ATP when oxidative phosphorylation in mitochondria is incapable of functioning. Hence, understanding how the Ldh mRNA is translated is important for cell metabolism, cell viability, and organismal physiology. Furthermore, discovering that a non-canonical form of eIF4E, called eIF4EHP in Drosophila, or eIF4E2 in humans, is responsible for this translation is an important finding that opens up new avenues for future research, asking more broadly which parts of the translatome depend on this factor for their translation. The fact that eIF4EHP knockdown flies fare less well than control flies in response to hypoxia shows that eIF4EHP is playing an important role.
• I think these findings will be interesting for a broad audience studying mRNA translation, hypoxia, cell stress responses, cell and organismal metabolism, and organismal physiology.
• My expertise is in Drosophila development, mRNA translation and tissue growth.
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Referee #1
Evidence, reproducibility and clarity
The manuscript by Gueydan and collaborators examines the role of eiF4EHP in Drosophila. The major conclusion of the paper is that eIF4EHP believed to be a translation repressor in fact drives protein synthesis during hypoxia. The bulk of the study is focused on the lactate dehydrogenase (LDH) mRNA, a mRNA that undergoes efficient translation during hypoxia thereby going against the grain of the general protein synthesis inhibition by low oxygen tension. While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism …
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Referee #1
Evidence, reproducibility and clarity
The manuscript by Gueydan and collaborators examines the role of eiF4EHP in Drosophila. The major conclusion of the paper is that eIF4EHP believed to be a translation repressor in fact drives protein synthesis during hypoxia. The bulk of the study is focused on the lactate dehydrogenase (LDH) mRNA, a mRNA that undergoes efficient translation during hypoxia thereby going against the grain of the general protein synthesis inhibition by low oxygen tension. While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism that adds what I consider to be another significant layer of evidence to the story. The paper also provides additional evidence that cells are equipped with multiple stimuli-specific cap-binding translation initiation factors that produce adaptive translatomes. The work is convincing, the paper well-written and the demonstration of eIF4EHP role in whole organism is important. Nonetheless, I do have a few comments that should be addressed to strengthen the conclusions of the authors.
The work focuses mainly of LDH. It would be a missed opportunity to show the effect of eIF4EHP in hypoxic Drosophila on puromycin incorporation. The work on S2 cells shown in Fig. 3E, while convincing, only reproduces what is already known. Puromycin incorporation in larvae.
In mammalian cells, blocking de novo transcription does not affect protein accumulation of eIF4E2 translated mRNA, even if these mRNA do not increase during hypoxia. It would be important to silence sima and test if this could block increased translation of ldh or other target in hypoxic conditions. This would suggest that sima is both a transcription and translation factor that evolved in HIF1a and HIF2a, the latter being a hypoxic translation regulator. This can be compared to cells treated with a general transcription inhibitor. These experiments would broaden the impact of the work
Fig 5C. It is unclear what are the conclusions of the authors are on the lack of co-localization between poly(A)RNA and eIF4EHP. In principle, eIF4EHP should co-localize with poly(A)mRNA. Perhaps a proximity ligation assay should be done to clarify this question. The data shown in F5C is not convincing one way or the other and needs clear cut experiments. Idem for 5A and B. PLA would provide convincing results.
Fig 6C is important but the data is hard to understand. First, the Y-axis is "enrichment relative to input". Is this hypoxia vs normoxia? Is the Y-axis log (probably)? The best would be to show that normoxia and hypoxia and see if eIF4EHP binds to ldh mRNA in both conditions perhaps acting as a translational repressor in normoxia and translational activator in hypoxia. Also, the authors could pool monosome and polysome factions and see in which fractions eIF4EHP binds to ldh mRNA. Finally, do the authors think that rpl32 remains associated with ldh mRNA in normoxia and hypoxia?
The authors should integrate the emerging concept of adaptive cap-binding translation factors in the discussion. It is still generally assumed that inhibition of eIF4E results automatically to cap-independent protein synthesis. The discovery that eIF3D, 4E2 and 4E3, amongst others, can initiate stimuli-specific translation should be discussed in the context of the work from this paper.
Significance
While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism that adds what I consider to be another significant layer of evidence to the story
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