PONE-D-25-00223Regulation of transcription elongation anticipates alternative gene expression strategies across the cell cyclePLOS ONE

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Reviewers' comments:

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Comments to the Author

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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

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

Reviewer #1: 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: No

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Reviewer #1: No

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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: A majority of studies dealing with understanding transcriptional regulation are focused on the initiation step. The transcription, however, is also regulated at the elongation and termination steps, and this downstream regulation is rather a neglected area of research. The focus of this investigation in elucidating regulation of transcription at the elongation step, therefore, is appropriate. Authors employ RNAPII ChIP and GRO approaches to study transcription at different stages of cell cycle in budding yeast. Their results suggest that genes coding for ribosomal components and linked to ribosome biogenesis are expressed at a higher level in pre-mitotic phases and are subjected to regulation at the elongation step. They also uncovered the role of mRNA stability in the overall expression of a number of these genes. These findings are significant and make an interesting scientific story. It is, however, difficult for a general reader of PLOS One to grasp and appreciate these results the way it is presented in this manuscript. Authors need to improve the presentation of results to make it more easily understandable. The following suggestions may help:

Major Points

1. Authors mention that some of the cycling genes are subjected to regulation at the elongation step. Inclusion of a list of these genes, and their RNAPII ChIP as well as GRO profile may help strengthen conclusions of the study.

2. Figure 1C is confusing. The X-axis shows time point after release from alpha-factor arrest, while Y-axis depicts Z-scores. It is, however, not clear what the headings on the top of every panel, G1, G1/S, S, S/G2, and M means. Does this mean the profile shown below is for genes expressed in the indicated stage of cell cycle? This needs to be clarified in the legend and also in the text. If G1 means the profile below is for genes predominantly expressed in G1 phase, then identity of these genes must be included in the form of a table in the supplementary data section.

3. In Figures 2B and S2B, authors need to explain in detail the basis of grouping genes in nine clusters. Just describing that clustering was done on the basis of RPCC and GRO profiles is not sufficient. Describe the range of RPCC and GRO signal values used for grouping genes in each cluster. Briefly explain SOTA while describing Figure 2A.

4. In Figure 2C, authors show distribution of gene clusters expressed in different stages of cell cycle. It will be beneficial for readers if these genes can be listed in the form of a table in supplementary data section.

Minor Points

1. In the third paragraph of ‘Introduction’, it needs to be clarified that TFIIS is not the only factor that regulates elongation. There are other factors, especially chromatin remodeling factors, histone chaperone and histone modifying enzymes that also regulate elongation through chromatin.

2. What is START? Explain for the benefit of readers not familiar with yeast.

3. Explain significance of R2 value.

4. In ‘Discussion’ section, briefly explain why mitochondrial related genes exhibit decreased mRNA stability with growth rate.

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Reviewer #1: No

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Regulation of transcription elongation anticipates alternative gene expression strategies across the cell cycle

PONE-D-25-00223R1

Dear Dr. Chávez,

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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Barbara Jennings

Academic Editor

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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

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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

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

Reviewer #1: Yes

**********

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

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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

**********

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: (No Response)

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

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