The Cap-Independent Translation of Survivin 5′UTR and HIV-1 IRES Sequences Is Inhibited by Oxidative Stress Produced by H. pylori Gamma-Glutamyl Transpeptidase Activity

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Rubilar et al. have submitted a manuscript entitled 'The cap-independent translation of Survivin 5'UTR and HIV-1 IRES sequences is inhibited by oxidative stress produced by H. pylori gamma-glutamyl transpeptidase activity.' The work focuses on demonstrating the presence of an IRES (Internal Ribosome Entry Site) in the 5' UTR region of the mRNA coding for Survivin, an anti-apoptotic protein.

The manuscript is a regular article and is a resubmission of a previous manuscript. In the previous revision phase, a series of comments regarding the discussion session were brought to the attention of the authors. In this new submission, the authors have implemented the discussion by satisfactorily responding to requests for revision.

Author Response

We sincerely thank the reviewer for their positive assessment of the revised manuscript. We are pleased that the improvements to the discussion section and our responses to previous comments were found to be satisfactory. We believe these revisions have significantly strengthened the manuscript and further clarified the relevance of our findings.

MV

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Thank you for carefully addressing my comments. However, the use of semicolons in lines 48-53 is unusual in standard English. Rather, I suggest that the lengthy sentence is broken up with periods

Comments on the Quality of English Language

See above

Author Response

We thank the reviewer for this helpful suggestion. The sentence in lines 48–53 has been revised by replacing semicolons with periods and breaking it into shorter sentences to improve clarity and readability.

MV

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a very interesting paper defining how Survivin is translated  as an anti-apoptotic protein primarily of the gut enterocyte and cancer biomarker.  There are many examples of internal ribosome entry mechanisms operating on endogenous mammalian genes such as that of VEGF mRNA. These are translational targets that are controlled like viruses, for example HIV in this study.  They provide a good in this paper to show that Survivin mRNA joins their ranks as presented in this paper.

They could make it clearer from the beginning how the shorter 5'UTR of the human survivin can be medicinally targeted to health benefit . Ie. small molecules that compete  with  H. pylori (likely 36 via GGT-induced oxidative stress) to offset  inhibition of IRES dependent translation of  Survivin (an anti apoptotic protein).  Please can the authors  make a clearer introduction to better introduce the clinical relevance of their result.

Their sentences were very long and it would be great to edit them toward clarity in this regard.

 

 

Comments on the Quality of English Language

Please edit the introduction with shorter sentences to give the readers a better immediate impression of this mechanism at work and its clinical relevance.  They focus on IRES-dependent survivin gene expression at the levels of message translation. 

Author Response

This is a very interesting paper defining how Survivin is translated  as an anti-apoptotic protein primarily of the gut enterocyte and cancer biomarker.  There are many examples of internal ribosome entry mechanisms operating on endogenous mammalian genes such as that of VEGF mRNA. These are translational targets that are controlled like viruses, for example HIV in this study.  They provide a good in this paper to show that Survivin mRNA joins their ranks as presented in this paper.

Response: We sincerely thank the reviewer for the positive and encouraging evaluation of our work. We appreciate the recognition of the novelty and biological relevance of identifying an IRES element in the Survivin mRNA, and we are pleased that the reviewer considers our results consistent with other well-characterized examples of IRES-mediated translation in mammalian genes.

They could make it clearer from the beginning how the shorter 5'UTR of the human survivin can be medicinally targeted to health benefit . Ie. small molecules that compete  with  H. pylori (likely 36 via GGT-induced oxidative stress) to offset  inhibition of IRES dependent translation of  Survivin (an anti apoptotic protein).  Please can the authors  make a clearer introduction to better introduce the clinical relevance of their result.

Response: We appreciate the reviewer’s valuable suggestion. We have revised the Introduction to better highlight the potential clinical implications of our findings. Specifically, we now emphasize that the short 5′UTR of Survivin may represent a novel therapeutic target, as modulation of its IRES-mediated translation could help restore Survivin expression and protect gastric cells from apoptosis under H. pylori-induced oxidative stress.

Their sentences were very long and it would be great to edit them toward clarity in this regard.

We thank the reviewer for this helpful observation. We have revised the Introduction to improve clarity and readability, shortening sentences and making the ideas more concise

 

Comments on the Quality of English Language

Please edit the introduction with shorter sentences to give the readers a better immediate impression of this mechanism at work and its clinical relevance.  They focus on IRES-dependent survivin gene expression at the levels of message translation.

Response: We thank the reviewer for this helpful observation. We have revised the Introduction to improve clarity and readability, shortening sentences and making the ideas more concise

 

Reviewer 2 Report

Comments and Suggestions for Authors

Rubilar et al. have submitted a manuscript entitled 'The cap-independent translation of Survivin 5’UTR and viral IRES sequences is inhibited by oxidative stress produced by H. pylori gamma-glutamyl transpeptidase activity.' The manuscript is a regular article.

The work focuses on demonstrating the presence of an IRES (Internal Ribosome Entry Site) in the 5’ UTR region of the mRNA coding for Survivin, an anti-apoptotic protein. The results reported in this article are a continuation of previous work in which the authors demonstrated that Survivin levels were decreased in human gastric tissue and cultured cells upon exposure to Helicobacter pylori gamma-glutamyl transpeptidase (GGT). The IRES characterization was performed excellently according to a well-accepted standard for demonstrating the existence of an IRES element, and the methodology was described with reasonable meticulousness in the Materials and Methods section.

The results show that the short 5’ UTR region of Survivin contains an IRES element capable of promoting the cap-independent translation of the corresponding messenger. Furthermore, confirming the results obtained in previous works, this IRES activity is inhibited following cell infection with H. pylori and/or ATO treatment, a known prooxidant.

The results are satisfactory and demonstrate the authors’ objectives with reasonable concordance. This finding, while running counter to a more general acceptance of the idea that cap-independent translation intervenes to sustain the synthesis of proteins crucial for cell survival in the presence of cellular stress, nevertheless finds similar analogies, described in the manuscript, where IRES activity is inhibited under similar conditions.

The discussion is quite complete, but I would like the authors to address the following points:

• Focus more on the significance of cellular IRESs, as extensive prominence was given to viral IRESs.
• The authors should explain at this point what advantage the presence of an IRES confers in the 5’ UTR region of Survivin.
• The authors should also explain whether, following the downregulation of survivin (consequent to the blockage of both cap-dependent and cap-independent translation), gastric cells might undergo apoptosis under stress conditions. Have the authors observed this phenomenon?
• How does the presence of the IRES in the survivin messenger fit into the context of a cancer cell? Could this actually be the mechanism through which Survivin expression is increased in tumors? Could it, therefore, be listed along with other anti-apoptotic genes, as highlighted in the recent work.
• A comment on the title: it refers to viral IRESs generically, but in the present work, the authors only analyzed the one from HIV. Please modify the title accordingly.
• Among all the results, I am not entirely convinced by the data obtained using siRNA silencing to evaluate the presence of other IRESs. As far as is known, siRNA silencing would lead to the complete degradation of the entire transcript. I am not sure if this data is truly essential, but above all, it is inconsistent with the function of siRNAs. The data suggests an uncharacterized mechanism, and therefore, in my opinion, it should be removed.
• In the figures, the word 'Luminiscence' is written; please correct it to 'Luminescence'."

Author Response

Comments and Suggestions for Authors

Rubilar et al. have submitted a manuscript entitled 'The cap-independent translation of Survivin 5’UTR and viral IRES sequences is inhibited by oxidative stress produced by H. pylori gamma-glutamyl transpeptidase activity.' The manuscript is a regular article.

 

The work focuses on demonstrating the presence of an IRES (Internal Ribosome Entry Site) in the 5’ UTR region of the mRNA coding for Survivin, an anti-apoptotic protein. The results reported in this article are a continuation of previous work in which the authors demonstrated that Survivin levels were decreased in human gastric tissue and cultured cells upon exposure to Helicobacter pylori gamma-glutamyl transpeptidase (GGT). The IRES characterization was performed excellently according to a well-accepted standard for demonstrating the existence of an IRES element, and the methodology was described with reasonable meticulousness in the Materials and Methods section.

Response: We thank the reviewer for the positive and encouraging comments regarding our work and the methodological rigor of the IRES characterization.

 

The results show that the short 5’ UTR region of Survivin contains an IRES element capable of promoting the cap-independent translation of the corresponding messenger. Furthermore, confirming the results obtained in previous works, this IRES activity is inhibited following cell infection with H. pylori and/or ATO treatment, a known prooxidant.

Response:  We thank the reviewer for the accurate summary and positive assessment of our results, as well as for recognizing the consistency with our previous findings.

The results are satisfactory and demonstrate the authors’ objectives with reasonable concordance. This finding, while running counter to a more general acceptance of the idea that cap-independent translation intervenes to sustain the synthesis of proteins crucial for cell survival in the presence of cellular stress, nevertheless finds similar analogies, described in the manuscript, where IRES activity is inhibited under similar conditions.

Response:  We thank the reviewer for the positive assessment and for the insightful interpretation of our findings. We agree that the observed inhibition of cap-independent translation under stress conditions may appear counterintuitive; however, as discussed in the manuscript, similar inhibitory effects on IRES activity have been reported in other contexts

 

The discussion is quite complete, but I would like the authors to address the following points:

 

Focus more on the significance of cellular IRESs, as extensive prominence was given to viral IRESs.

Rsponse: We have revised the Discussion to emphasize this point and to further elaborate on possible mechanisms underlying this phenomenon.

 

The authors should explain at this point what advantage the presence of an IRES confers in the 5’ UTR region of Survivin.

Response: We thank the reviewer for this relevant comment. We have now added an explanation in the Discussion section regarding the potential advantage conferred by the presence of an IRES in the Survivin 5′UTR.

The authors should also explain whether, following the downregulation of survivin (consequent to the blockage of both cap-dependent and cap-independent translation), gastric cells might undergo apoptosis under stress conditions. Have the authors observed this phenomenon?.

Response: Indeed, in our previous work , we observed that H. pylori infection led to a marked decrease in Survivin levels in gastric epithelial cells, which correlated with the activation of apoptotic markers and increased cell death. These findings suggest that Survivin downregulation promotes apoptosis under stress conditions.  We have added a brief explanation and a citation to our previous study in the revised Discussion to clarify this point.

How does the presence of the IRES in the survivin messenger fit into the context of a cancer cell? Could this actually be the mechanism through which Survivin expression is increased in tumors? Could it, therefore, be listed along with other anti-apoptotic genes, as highlighted in the recent work.

Response: We agree that the presence of an IRES in the Survivin 5′UTR could contribute to its overexpression in cancer cells, where global cap-dependent translation is often impaired due to stress or metabolic imbalance. Under such conditions, IRES-mediated translation could ensure sustained Survivin synthesis, supporting cell survival and resistance to apoptosis. We have expanded the Discussion to include this point and to relate Survivin to other anti-apoptotic genes that may employ similar regulatory mechanisms, as highlighted in recent studies.

A comment on the title: it refers to viral IRESs generically, but in the present work, the authors only analyzed the one from HIV. Please modify the title accordingly.

Response: We appreciate the reviewer's helpful observation. We agree that the title should be more specific and have modified it accordingly to refer explicitly to the HIV IRES.

Among all the results, I am not entirely convinced by the data obtained using siRNA silencing to evaluate the presence of other IRESs. As far as is known, siRNA silencing would lead to the complete degradation of the entire transcript. I am not sure if this data is truly essential, but above all, it is inconsistent with the function of siRNAs. The data suggests an uncharacterized mechanism, and therefore, in my opinion, it should be removed.

We thank the reviewer for this thoughtful and constructive comment. We agree that siRNA silencing has intrinsic limitations, as it may lead to degradation of the entire transcript. However, we followed the methodology described in two previously published studies (in Journal of Virology and Nucleic Acids Research), where a similar approach was used to assess the potential involvement of IRES elements. While we acknowledge the constraints of this method, these data, when considered together with the rest of our results, reinforce the general idea of our work. We have clarified this point and included the corresponding references in the revised version of the manuscript.

 

In the figures, the word 'Luminiscence' is written; please correct it to 'Luminescence'."

Response: We thank the reviewer for noticing this typographical error. It has been corrected in all the corresponding figures in the revised version of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

In the current manuscript (biomolecules-3929453), Rubilar et al present experiments aimed at evaluating promoter and translation initiation sites in two versions of the 5’UTR of Survivin. Their results suggests that the long version (UTR_L) harbors a cryptic promoter, while both the  UTR_L and short UTR (UTR_S) contain cap-independent translation initiation sites). Their results further suggest that gamma-glutamyl transpeptidase (GGT) activity of H. pylori inhibition of cap-independent translation initiation may be a global phenomenon. The experiments are generally well-designed and executed according to the standards in the field. The most serious problem with the manuscript is the meandering style and the failure to clear define the in vivo observations that motivated the current experiments.

 

Major issues

1. Although the authors sporadically refer to relevant in vivo observations, mostly reported by their own group, they never give a concise definition of the phenomenon for which they seek a mechanical understanding. This is perhaps most evident from the diffuse wording of the conclusion in lines 647-651).
2. Please define all abbreviations.
3. Lines 44-50 are one extremely long sentence that goes on and on. Break it up, please.
4. Lines 117-127: Rewrite to define more precisely the question you are trying to answer.
5. Figure 1E, lane 2: HIV IRES mRNA migrates slower that other mRNAs, even though Figure 1A indicates that the UTR_L mRNA is the longest transcript. Why?
6. Figure 1A: Explain colors.
7. Figure 2C, HEK293 panel. Why are no stats associated with the UTR_S pSV40-minus Fluc column?
8. Lines 377-379 should be moved to the discussion.
9. Line 394: verify by mutating the AUG.

Author Response

Comments and Suggestions for Authors

In the current manuscript (biomolecules-3929453), Rubilar et al present experiments aimed at evaluating promoter and translation initiation sites in two versions of the 5’UTR of Survivin. Their results suggests that the long version (UTR_L) harbors a cryptic promoter, while both the  UTR_L and short UTR (UTR_S) contain cap-independent translation initiation sites). Their results further suggest that gamma-glutamyl transpeptidase (GGT) activity of H. pylori inhibition of cap-independent translation initiation may be a global phenomenon. The experiments are generally well-designed and executed according to the standards in the field. The most serious problem with the manuscript is the meandering style and the failure to clear define the in vivo observations that motivated the current experiments.

Major issues

1. Although the authors sporadically refer to relevant in vivo observations, mostly reported by their own group, they never give a concise definition of the phenomenon for which they seek a mechanical understanding. This is perhaps most evident from the diffuse wording of the conclusion in lines 647-651).

Response:We thank the reviewer for this valuable and constructive comment. We have considered this observation carefully and have revised both the Introduction and the Discussion to more clearly define the biological phenomenon that our study aims to elucidate. In addition, we have rewritten the Conclusion (lines 647–651 in the previous version) to make our main message more explicit and better aligned with the experimental findings.

 

2. Please define all abbreviations. We have defined all abbreviations that were not previously defined throughout the manuscript.

 

3. Lines 44-50 are one extremely long sentence that goes on and on. Break it up, please.The text has been revised to improve clarity and readability.

 

4. Lines 117-127: Rewrite to define more precisely the question you are trying to answer.

We appreciate the reviewer’s valuable suggestion. Accordingly, the text has been rewritten to more clearly define the research question addressed in this study. The revised version now specifies that our work aims to determine how Helicobacter pylori infection regulates Survivin expression at the translational level under stress conditions through mechanisms involving the 5′UTR.

 

5. Figure 1E, lane 2: HIV IRES mRNA migrates slower that other mRNAs, even though Figure 1A indicates that the UTR_LmRNA is the longest transcript. Why?

Response: We thank the reviewer for noticing this detail. The apparent discrepancy was due to a minor artwork alignment issue in the original figure. This has been corrected in the revised version, and the migration pattern now accurately reflects the expected transcript sizes

 

 

6. Figure 1A: Explain colors.

Response: We thank the reviewer for this useful comment. The colors used in the plasmid schematic (green, orange, and yellow) were selected arbitrarily to visually differentiate genes and regulatory elements. We have revised the figure to ensure that the same color scheme is applied consistently across all figures in the manuscript.

 

 

7. Figure 2C, HEK293 panel. Why are no stats associated with the UTR_SpSV40-minus Fluc column?

 

Response: We thank the reviewer for this observation. We thank the reviewer for this observation. The lack of statistical information in the UTRS pSV40-minus Fluc column was due to an unintentional displacement of the error column when the figure was originally generated in Excel. The figure has now been corrected and recreated using GraphPad Prism to ensure accuracy and proper alignment of all data and error bars.

 

 

Lines 377-379 should be moved to the discussion.

Response: We agree with the reviewer. The indicated sentences have been moved to the Discussion section accordingly.

 

 

8. Line 394: verify by mutating the AUG.

Response: We appreciate the reviewer’s insightful suggestion. While mutating the AUG start codon would indeed provide additional evidence to confirm this mechanism, such an experiment was beyond the scope of the current study. However, we acknowledge this limitation in the revised version and mention that future work will include mutational analysis of the AUG to validate this hypothesis.