PONE-D-25-01723Beyond the ORF: Paralog-specific regulation of RPS7/eS7 mRNAs via 3'-UTRs and promoter sequences

PLOS ONE

Dear Dr. Hayashi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Please submit a revised manuscript after addressing these comments. Additionally, ensure that your revision includes a discussion of any potential limitations in the experimental design or study.

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Koppolu Raja Rajesh Kumar, PhD

Academic Editor

PLOS ONE

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“This work was supported by JSPS KAKENHI (https://www.jsps.go.jp/english/) Grant number JP20K06491 (to S. H), 17H05672 and 23K18100 (to T. Y.), Takeda Science Foundation (https://www.takeda-sci.or.jp/en/), and Hyogo Science and Technology Association (https://hyogosta.jp/), Japan (to S. H.; 6082). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The work of Hayashi and Yoshihisa is a demonstration of the use of scientific reasoning to investigate the molecular mechanisms of life from a serendipitous finding. The work contains many results, with varying relevance. Some of them serve to interpret the phenotype of the spontaneous mutant, which has little biological relevance, and others focus on the study of a new mechanism of cross-control between paralogous copies of a yeast ribosomal protein.

1) On page 13, line 194, the mRNA of the CaHIS3 insertion version in Fig. 2C is longer than wt. This should be mentioned in the text.

2) On page 13, line 199, the Rps7A protein level in wt is not shown. This is because wt is not HA-tagged, but it would be interesting to have a relative quantification of Rps7A levels in Rps7A-CgHIS3 with respect to those in wt.

3) Page 15, line 228. There are two ... at the end of the line.

4) Page 17, line 268. “we deleted this sequence...”. It is important to know which sequence was deleted. Since there is a putative 5 bp overlap between the Rap1 and Fhl1 binding sites, if the entire sequence is deleted the consequence would be a lack of both binding sites. If only part of it is removed, Rap1 (or Fhl1) may still bind to it. According to line 272 on page 18 both sites were removed but this conflicts with other conclusions given later. See point #5 below.

5) Line 272 states that (Fig. 5B) the lack of both Rap1/Fhl1 binding sites is not affecting Rps7A expression (as demonstrated by the GFP reporter). This is quite surprising because these factors are described as very important for RP gene expression in many previous papers. Here it appears that in the RPS7A gene they are only required for negative feedback of the Rps7 protein on RPS7A transcription. Although the question is discussed below (pages 23-24) it is quite surprising and as no potential mechanism is suggested it leaves the question too unresolved. Is the Rps7 protein interacting with Rap1 (or Fhl1, or another factor) to prevent binding? If so, the experiment shown below (fhl1 mutant) will demonstrate that Fhl1 is NOT the target of Rps7A. As for the function of Rap1, since Rap1 is essential a ts mutant or Auxin-degron can be used to test the function of Rap1 in Rps7A transcription. Alternatively the rap1delta-sil mutant has been used in the context of RP gene expression where it affects the RNA pol II transition between initiation and elongation (doi:10.1371/journal.pgen.1000614).

6) page 24 line 373. RPS7 is RPS7B?

7) In Discussion (page 24) the authors describe the existence of other TFs that can be activating Rps7A promoter, such as Ifh1 or Hmo1. I think that Sfp1 should be included because it another well-known transactivator of many RP genes.

Reviewer #2: The authors focused on a very interesting topic: what does it happen with paralogous genes? how redundant/specific are they? They have focused on a pair of ribosomal proteins (RPS7A and RPS7B). The main problem of the paper is that the main aim of the study, the specific objectives and the hypotheses are never clearly stated upfront. Therefore, the reader does not know if the described experiments make sense in order to achieve that aim and answer those research questions.

In my opinion, some of the presented experiments seem irrelevant in order to understand the relationship between RPS7A and RPS7B. For example, the authors constructed a strain with an artificially long 3'UTR in RPS7B. Then, they demonstrate that NMD controls the expression of that modified RPS7B gene. However, I wonder why this experiment is relevant if this never occurs in natural/biotechnological strains. WT RPS7B has a normal 3'UTR which is probably not targeted by NMD. Therefore, the implications of these results are totally obscure to me.

Moreover, other comparisons do not seem to be absolutely fair. In my opinion, it is very interesting to know how the amount of mRNA and transcript of one of the paralogs is affected when the other paralog is knocked down or overexpressed. Here, the authors have looked at the effect of the RPS7A deletion on RPS7B. They have also looked at the effect of the RPS7B deletion on RPS7A. However, most of the quantitative analyses on RPS7A where done with the long-UTR RPS7B gene. In this experiment, the transcription of RPS7B is not prevented, but the mRNA is mostly degraded and hence never translated. The reciprocal experiment (RPS7A targeted by NMD) is never done. In order to rule out symmetrical effects, the analyses need to comprehensive.

Finally, I am not entirely satisfied by the way that the statistical analyses are performed, reported and interpreted.

1) I did not see information on the number of replicates used in each experiment; n must be reported.

2) t-test is used in most analyses, but this is incorrect because many of those involve multiple comparisons. If all comparisons are of interest, the authors should use ANOVA plus a post hoc test (e.g. Tukey's test). If only comparisons to a single control are of interest, Dunnet's test should be used instead. If not all the comparisons are of interest and there is no common control, the authors should correct the p-values of the t-test for multiple testing (because they are interested in any significant difference, their type-I error is above 0.05).

3) the authors disregarded an interesting result because it does not agree with their main conclusion (RPS7B expression depends on RPS7A expression, but RPS7A expression is not affected by RPS7B). When describing Figure 1B, the authors state "rps7bΔ affected RPS7A expression only slightly (rps7bΔ/WT, 0.70±0.13; by Student’s t test, p = 0.0066)" in lines 177-178. I totally disagree with this statement. First, the difference is either statistically significant or not; in this case, it clearly is (p-value < 0.01). Slightly is not a statistical term. Second, RPS7A expression is clearly depleted in the rps7bΔ strain; it is just 70% of the expression in the WT strain. A 30% depletion is not a slight change. This result should be taken into account when interpreting the rest of analyses.

For all these reasons, I have to recommend that the paper is rejected. I would suggest to the authors that they decided on what is the main aim of their research and then they did a complete rewriting of the paper focusing on achieving that aim.

Reviewer #3: Hayashi and Youshihisa present their results from investigating the regulation of the yeast RPS7A and B paralogs. Because the yeast genome resulted from a whole-genome duplication event, many proteins are duplicated (termed ohnologs). This includes many ribosomal proteins. Previous work has reported that the alternative copies of ribosomal proteins present in the yeast genome have different regulation and, potentially, somewhat different functions. In this manuscript, the authors report similarities and differences in the regulation of the expression for ohnologs RPS7A and RPS7B. In particular, they report that the short 3' UTR of both genes is important for RNA stability, and that the transcriptional regulation of both RPS7A and 7B respond to a decrease in RPS7A expression, suggesting a feedback loop. The authors also describe some differences in the functions of transcription factor binding sites upstream of the two genes. Overall, this is a well performed study that is interesting to the gene regulation and ribosome research communities. I have mostly minor comments that should be addressed:

1. It has been reported that deletions of ribosomal proteins can cause yeast to become aneuploid as a response to loss of the RP copy (see PMID: 30503772 and PMID: 22377630). To elaborate, segments or entire chromosomes carrying the RP copy that is still present will duplicate, as cells that have the "normal" number of copies are more fit than those that lose one copy (even with the aneuploidy). The authors should note this in the introduction and discussion, as it is relevant to their observation that RPS7A deletion leads to an increase in RPS7B mRNA. It is possible that these increases could be due, at least in part, to amplification of the remaining RPS7 DNA copy.

2. Line 73. What does "delicate to" mean? I don't understand.

3. Line 90 "gene region with an amplified" -> "gene region was replaced with an amplified"

4. Line 192 "phenotype appeared" -> "phenotype that appeared"

5. Line 214 to 215 is confusing. Perhaps try, "Unlike tl(caa)deltaint*, neither rps7b-101 nor rps7b-102 conferred any growth defect at 30C on YPD.

6. Line 273 "unchanged" -> "did not change"

7. Line 304, is that a period after strain? should be comma?

8. Lines 325-326 are confusing.

9. Line 360 "supposed" -> "expected"

10. Line 390 "dependet" -> "dependent"

11. Line 399 " preference for RPS7Bp". The paper referenced shows a preference for RPS7A, not B in higher protein level.

12. Line 406 "demonstrates" -> "suggests". demonstrates is a bit too strong.

13. Line 407 "mRNA level" -> the surveillance or feedback is probably on the protein level or in response to the amount of assembled ribosomes. I don't think there's any evidence that the actual mRNA levels is directly sensed (again why I think it's not "demonstrated"; see comment 12).

14. RPS7A mRNA (probably protein, not mRNA). Most likely mechanism is a problem in ribosome assembly, with ribosomes getting stuck in particular stages of assembly. I wonder if there are feedback mechanisms that turn on expression of different genes when different subunits are not assembled well.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-25-01723R1Beyond the ORF: Paralog-specific regulation of RPS7/eS7 mRNAs via 3'-UTRs and promoter sequencesPLOS ONE

Dear Dr. Hayashi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Reviewer 3 has raised a specific concern that must be addressed before the manuscript can be accepted for publication.

I kindly request you to respond to the comment(s) and resubmit the revised manuscript.

I look forward to receiving your revised submission.

==============================

Please submit your revised manuscript by Jun 08 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Koppolu Raja Rajesh Kumar, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All questions have been solved or argued. The paper can be published in the current version in PLOS One

Reviewer #3: The authors' revision is much improved. I only have one request. As I mentioned in the first review, it is technically possible that the increase in RPS7B RNA levels that occurs upon deletion of RPS7A could reflect duplication of the chromosome on which RPS7B is found (aneuploidy). The authors should explicitly acknowledge that in the discussion. Currently, they only vaguely hint at the possibility.

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Reviewer #1: No

Reviewer #3: No

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Beyond the ORF: Paralog-specific regulation of RPS7/eS7 mRNAs via 3'-UTRs and promoter sequences

PONE-D-25-01723R2

Dear Dr. Hayashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Koppolu Raja Rajesh Kumar, PhD

Academic Editor

PLOS ONE

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