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PONE-D-24-40299The RNA-binding protein Puf5 and the HMGB protein Ixr1 regulate cell cycle-specific expression of CLB1 and CLB2 in Saccharomyces cerevisiaePLOS ONE

Dear Dr. Irie,

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.

Both reviewers raise substantial editorial and technical issues and consider that some of your conclusions are not backed up by the experimental evidence that you present. These include the evaluation of the protein expression levels of Clb1 and Clb2 as well as direct measurement of DNA damage. We invite you to submit a revised version of the manuscript that addresses all the points listed below (see the reviewers' reports for more details):

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Academic Editor

PLOS ONE

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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

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

Reviewer #1: Yes

Reviewer #2: 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: Yes

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4. 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 #2: 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: In this manuscript, the authors reported that the RNA-binding protein Puf5 regulates the expression of CLB1, as previously reported, and also CLB2, albeit to a lesser extent. This regulation of both CLB1 and CLB2 by the Puf5-Ixr1 machinery involves the Dun1 kinase. Additionally, it was observed that the G2-M cyclins, Clb1 and Clb2, have overlapping functions with the S-phase cyclins, Clb5 and Clb6.　

While some potentially interesting results have been observed, some minor corrections are desirable before publication.

1. Please provide the number of experiments conducted or the biological replicates for Figures 3, 4, 5, and 6 in the figure legends.

2. The authors should carefully review the entire text.

Introduction, line 131: The phrase “the arrest of the cell cycle at G2/M phase arrest” contains redundant wording.

Results, line 168: The word "consistently" should be reconsidered. It seems a bit of over-speculation in this context.

Lines 177 and 178: “the expression…CLB5 and CLB6 remained unchanged”. According to Supplemental Figure 1, the expressions of CLB5 and CLB6 were slightly down-regulated, and their expression timing was shifted to later.

Lines 179 and 180: “CLB2 expression was preserved in the puf5Δ mutant”. This contradicts the section title “Expression of CLB2 is reduced in the puf5Δ mutant.”

Lines 209 and 210: “The clb1Δ clb5Δ clb6Δ triple …than the wild-type strain.” There is no wild-type spore in Figure 2B.

Line 213: There is a “-” between the sentences.

Lines 243 and 244: “the puf5Δ clb5Δ double…by the multi-copy CLB2, CLB5, and CLB6”. In Figure 9A, complementation with YEpCLB6 at 37°C was not sufficient.

Lines 269 and 270: “a single copy or…inhibited the growth of the ixr1∆ dun1∆ clb2∆”. The suppression of growth by a single copy CLB2 plasmid was very mild (Figure 10B).

Line 286: The phrase “like the sml1Δ mutation” is a bit confusing, as the authors mentioned earlier in lines 279 and 280 that “the effect by … is different from the effect of the sml1Δ deletion.”

Lines 290: “This is consistent with” sounds too conclusive; it might be better to soften the statement.

Lines 298 and 299: “the dun1Δ clb6Δ double…slow growth”. It is difficult to see the slow-growth phenotype of that mutant (Figure 13A).

Lines 306-310: I think there is a missing “,” in the sentence that begins with “Although a significant...”.

Lines 313: “was may be” is not correct.

Lines 317-319: The last sentence of the results section is purely speculative, so it might be better to move it to the discussion section.

Discussion, line 330: “a clear decrease” should be softened.

Lines 374-381: The sentences may need to be reviewed for clarity regarding the author’s claim.

Reviewer #2: This manuscript by Sato et al. builds upon their previous findings on the regulation of CLB1 expression by the RNA-binding protein Puf5 in yeast genetics. The authors extend their investigation into the broader impact of Puf5 on the cyclins. By using synchronized cell cultures, they reveal that Puf5 regulates not only CLB1 but also CLB2, and suggest that this regulation may be mediated by the HMGB protein Ixr1. Furthermore, they demonstrate that Clb2 can compensate for the functions of Clb5 and Clb6. Their study highlights the significance of the Puf5-Ixr1 mechanism in the fine-tuned regulation of the cell cycle through cyclin gene expression control. However, several issues need to be addressed before publication in PLOS ONE.

<major comments="">

The study proposes a mechanism for Puf5's regulation of the cell cycle by demonstrating the degree of yeast growth and qRT-PCR results using genetics. However, it remains unclear whether the expression levels of cyclin mRNA and cyclin proteins are correlated. To strengthen the validity of the study, it would be helpful to show protein expression levels of Clb1 and Clb2, particularly in Figure 1 and Figures 3C, D at the 60-80 min time points, which are critical to the study.

Lines 218-221: The delay in SIC1 mRNA expression in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant is attributed to a G2 phase delay, but it is also possible that cell cycle synchronization is inconsistent. It would be advisable to soften the expression in the manuscript or support the authors' conclusions by performing nuclear and cell wall staining followed by microscopic observations.

Lines 225-227: Both CLB2 overexpression and PUF5 overexpression suppress the growth defect of the puf5Δ clb1Δ clb5Δ clb6Δ strain, but PUF5 overexpression appears to promote faster growth. Could this be due to PUF5 overexpression regulating target molecules other than CLB2? Alternatively, when using a plasmid, the cell cycle-dependent expression of Clb2 may not be fully replicated, and plasmid-derived Clb2 might not function as effectively as endogenous Clb2. Since PUF5 overexpression regulates endogenous Clb2 expression, it could restore the cell cycle-dependent expression pattern of Clb2, leading to better recovery of Clb2 function than CLB2 overexpression. Current data do not clarify whether CLB2 expressed from a plasmid exhibits cell cycle-dependent expression, making it difficult to determine whether CLB2 overexpression is fully functional. Including clb1Δ clb5Δ clb6Δ-YEplac195 as a control would allow for an evaluation of whether the effects of exogenously expressed CLB2 overexpression are complete or partial.

Lines 265-266: Does CLB1 play a role in the growth defect of ixr1Δ dun1Δ? Would the ixr1Δ dun1Δ clb1Δ clb2Δ strain show improved growth compared to the triple mutant, ixr1Δ dun1Δ clb2Δ? To discuss the functional distinction between Clb1 and Clb2, it would be preferable to show the effects of clb2Δ on ixr1Δ dun1Δ.

Lines 288-291: DNA damage is being indirectly assessed by RNR3 expression. It would be preferable to directly evaluate DNA damage. If not, it may be better to move this statement to the Discussion section. Since there are many repeated sections in the Results and Discussion, combining these sections into one as "Results and Discussion" might be more effective.

Lines 297-299: In Figure 13A, the growth of dun1Δ clb5Δ appears slow, but dun1Δ clb6Δ shows little change in growth compared to dun1Δ and clb6Δ.

Lines 352-353: In Figure 9A, CLB1 overexpression does not appear to suppress the growth defect of puf5Δ clb5Δ. Thus, CLB1 may not compensate for CLB5. It would be important to examine whether CLB1 overexpression can suppress the growth inhibition of puf5Δ clb5Δ clb6Δ. Alternatively, the effect of CLB1 overexpression on the growth and temperature sensitivity of puf5Δ clb5Δ should be more accurately evaluated.

Why can Clb2 compensate for Clb5 and Clb6? A discussion of the physiological significance of this compensation would be desirable.

<minor comments="">

The figures appear somewhat disorganized, so it would be better to reconsider the integration and order of the figures to match the manuscript.

Line 204: The statement that the puf5Δ clb3Δ clb4Δ triple mutant did not show any growth delay could be softened, as Supplemental Figure 2 seems to show a slight growth retardation. A phrase like "showed slight growth retardation" would be preferable.

An explanation of what DUN1 is should be provided not only in the abstract but also in the main text, particularly in the Results section.

The notation of genotypes should be consistent between the manuscript and the figures. For example, in the manuscript, clb1Δ clb5Δ clb6Δ is used, but in Figure 6, clb5Δ clb1Δ clb6Δ is written.

Figures 4A and B are not explained in the manuscript.

Are the cells in Figure 6 in a bar1Δ background? If so, the description in both the figure and the manuscript should be revised accordingly.</minor></major>

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Reviewer #1: No

Reviewer #2: No

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The RNA-binding protein Puf5 and the HMGB protein Ixr1 regulate cell cycle-specific expression of CLB1 and CLB2 in Saccharomyces cerevisiae

PONE-D-24-40299R1

Dear Dr. Irie,

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.

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Kind regards,

Reiko Sugiura, M.D., PhD.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

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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 #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: 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: The authors have addressed all the points raised by this reviewer in the initial version of the manuscript.

Reviewer #2: The authors have responded to the reviewers' comments with integrity, conducted appropriate additional experiments, and provided clear explanations. The revised manuscript is deemed to have sufficient merit for publication in PLOS ONE.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Ryosuke Satoh

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