PONE-D-22-27372Telomerase RNA plays a major role in the completion of the life cycle in Ustilago maydis and shares conserved domains with other UstilaginalesPLOS ONE

Dear Dr. Sanchez-Alonso,

Thank you for resubmitting 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.

An additional issue has been raised by  Reviewer 2 that was not addressed in a previous critiques. Specifically is not fully clear that that the gene identified does indeed encode the telomerase RNA. This should be accomplished, if technically feasible, by a simple complementation assay. In the absence of this information, a qualification of the conclusion must be made within the paper and in the title.

In addition to this experimental change, please address minor issues raised by the second reviewer.​

Please submit your revised manuscript by Jan 05 2023 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,

Arthur J. Lustig, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

5. 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.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: This revised manuscript reports the identification and characterization of the gene encoding telomerase RNA from Ustilago maydis, a basidiomycete fungus. The RNA is evidently synthesized from an upstream ORF (UMAG_03168), and subjected to splicing and processing to generate the mature form. Disruption of the telomerase RNA gene results in telomere shortening and reduced proliferation. The ter1 mutant is also defective in several aspects of the U. maydis life cycle. The authors identified conserved features of the RNA, including potential pseudoknots and Sm-binding sites.

The discovery of telomerase RNA from a basidiomycete fungus is an important advance and will pave the way for structural, functional and comparative analysis of this conserved lncRNA. The main concern for the original manuscript was the lack of strong supporting data for the proposed mechanism of RNA biogenesis involving transcription from an upstream protein coding RNA. The authors have now provided RT-PCR analysis that are consistent with the proposed mechanism. They also made additional changes to address other concerns. I believe these changes have substantially strengthened the manuscript.

It is worth noting that during the revision, the Julian Chen lab published a PNAS paper that describes their characterization of the same telomerase RNA. I think the two studies are complementary. The Chen paper provides a very thorough and convincing characterization of the biogenesis mechanism that leaves very little doubt about the nature of the precursor as a polycistronic RNA. The current manuscript, on the other hand, provides interesting data on the role of this RNA in vivo; the telomere lengths analysis of the ter1 mutant is more convincing and the role of this RNA in the life cycle of U. maydis is a nice finding. In short, I think this is a solid manuscript that helps to advance the field.

Reviewer #2: Regarding the manuscript titled "Telomerase RNA plays a major role in the completion of the life cycle in Ustilago

maydis and shares conserved domains with other Ustilaginales" by Sanchez-Alonso et al. I think the authors have appropriately addressed the concerns of the previous reviewers however I have one concern and the editor will need to determine if it must be carried out before publication This is, that authors did not confirm that the deletion they carried out was responsible for the observed phenotypes. Generally a deletion mutant is complemented by adding back the gene at an ectopic locations. In this case the authors would need to add back the RNA and determine if doing so complements the deletion phenotypes observed. This could be done using genome integration or an autonomously replicating plasmid and assessing the haploid cell phenotypes. If this is not done i think the title should be modified to indicate that the gene identified is a probable or predicted telomerase RNA.

Other minor points the authors may consider addressing are wording issues and matters of sentence clarity. I have highlighted some below

Lines 117-121 Clarify, when does the repression of the putative TER gene occur perhaps break the sentence into two to help clarify the points being presented

Line 126 the phrase "order representatives here studied" is awkward and not clear, edit for clarification

Lines 237-239 After running the sucrose cuchion centrifugation were samples assessed to determine the purity and degree of subcellular fractionation, ie how pure were the nuclei isolated in this manner?

Lines 550-554 The portion of the sentence beginning "ratifying ..." is not clear and could be edited to improve clarity. Also consider breaking up this overly long sentence

Lines 581-582 "results points to the fact" should be edited to results suggest or results support an RNA maturtion process that involves

Lines 583-586 The portion of the sentence " ...,so unlike TLC1... " seems like discussion not results. Consider moving this to the discussion.

Line 612 the phrase "every at approximately 24 duplication rounds, " is not clear edit to clarify what you intend to convey

Line 649 and elsewhere The phenotypic differences that distinct mutants present lead me to ask - Were there any DNA-level differences detected between the mutants that might help explain mutant to mutant variation?

Line 687 has should be had

Line 751 should " extended a pair" be extended by a pair?

Lines 752-754 wording of this sentence could be improved eg denotate does not seem to be a good word choice here

Lines 757-759 the sentence beginning "Whether ..." is not clear and could be edited to improve clarity

Lines 796 -798 This sentence is not clear edit to better explain what you are trying to convey

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

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: Barry Saville

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Telomerase RNA plays a major role in the completion of the life cycle in Ustilago maydis and shares conserved domains with other Ustilaginales

PONE-D-22-27372R1

Dear Dr. Sanchez-Alonso,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arthur J. Lustig, 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 #2: (No Response)

**********

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 #2: Yes

**********

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

Reviewer #2: 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 #2: Yes

**********

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 #2: 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 #2: I would like to thank the authors for the respectful response to my initial concerns regarding the manuscript. While I accept that the ter1Δ complementation would be unfeasible in the traditional sense, that is growing up the mutant and adding back the wildtype gene, the authors should consider a different approach. Perhaps in a follow on experiment, this approach is a two step deletion in which they insert a wildtype copy of the locus to be deleted into the IP locus under the control of a promoter that can be controlled eg by carbon source then they delete the wildtype copy of the gene and grow out the deletion mutants with hyg selection and under permissive conditions expressing the ectopically inserted gene. Subsequently they can double spot the double transformants under permissive and non-permissive conditions and assess the impact on phenotypes previously assessed. We have gotten this to work with genes that are lethal when deleted. The advantage of considering this is it would allow mutational analysis of the ectopically expressed copy. I think this might take some time and I think it may open up the possibility for future investigations so I would not consider this a requirement for publication of this work, but it may be interesting for future analyses.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

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 #2: Yes: Barry J Saville

**********