Dear Dr Palanisamy,

Thank you very much for submitting your Research Article entitled 'RNA Binding Protein FXR1-miR301a-3p axis contributes to p21WAF1 degradation in oral cancer' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a much-revised version. We cannot, of course, promise publication at that time.

While all authors recognize the novelty, innovation, and importance of the subject, there were concerns with the inclusion of appropriate controls for the EMSA and other experiments.  It needs to be determined if FXR1 directly alters PNPT1 to determine effects on miRNA degradation, performance of experiments in the presence of transcription inhibitors, and a direct binding of PNPT1 to miR301.  It is also strongly recommended to clarify the binding site proximity for FXR1 and miR301 in the p21 3'UTR.  Finally, Reviewer #2 brings up a good point in that cellular experiments should be performed to validate the proposed molecular axis.  This would add physiological validity to the model.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see our guidelines.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Michael V. Autieri

Guest Editor

PLOS Genetics

David Kwiatkowski

Section Editor: Cancer Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In this manuscript, the authors reported that a RNA-binding protein FXR1 control miR301a-3p stability by directly binding to this miRNA and prevent its degradation by PNPT1 in HNSCC. As miR301a targets p21, thereby FXR1 promote the growth and proliferation of HNSCC. The report does contain some innovative findings. The manuscript is well written and all of data are beautifully presented. However, all of data were collected on the cultured cell lines. No data show there is functional relevance in vivo. The reviewer think the report is still preliminary and need to add some in vivo observation.

Reviewer #2: The manuscript “RNA binding protein FXR1-miR301a-3p axis contributes to p21WAF1 degradation in oral cancer” provides a compelling case that FXR1 protects miR301a-3p from degradation from exonuclease PNPT1 which allows the miR to destabilize p21, forming a novel axis of regulation in oral cancer cells. Overall, I find this manuscript to be of great interest to the fields of both RBP-miR interactions and RNA regulation of cancer biology.

Major Critiques:

1. Figure 2A needs a negative control gene in addition to the IgG control shown for FXR1-miR 301a-3p interaction by pulldown.

2. Figure 2 shows an EMSA gel shift experiment, but the figure would be more convincing and enhanced using a negative control probe to demonstrate specificity in FXR1 binding in vitro.

3. Figure 4E again would be enhanced by a negative control probe especially due to degradation of miR301a-3p probe by PNPT1 to again show specificity of the interaction and degradation.

4. Although it is clear FXR1 binds miR301a-3p, can you rule out that FXR1 directly alters PNPT1 to directly or indirectly prevent miR degradation?

5. The overall interaction axis between FXR1-miR301a-3p-p21 suggests effects on cellular senescence and proliferation, and experiments measuring changes in the cell would strengthen the paper demonstrating tangible effects of this interaction in cancer cells. These cellular experiments should be performed manipulating participating components of the novel axis to determine effects on cellular function.

Minor Critiques:

1. The paper introduces and shows data involving the FXR1-miR29b-3p interaction but fails to explain why this was not continued.

2. Discussion points: Does miR 301a-3p target any other cancer or senescence related transcripts other than p21 based on the target sequence? Further, does FXR1 bind p21 directly and regulate the transcript by RNA stability, and how that might affect the role of FXR1 in the axis identified.

Reviewer #3: Majumder and Palanisamy describe a novel and very relevant regulatory model involving miR-301a and the RNA binding protein FXR1. The data is convincing and supports their model. However, there are a few weak points and further analysis should be done to expand their conclusions and increase the value of their article.

Major points

-Changes in transcription levels could interfere with results. Transcription should be shut down, then they can properly evaluate the impact of FXR1 on miR-301a stability.

-In Figure 2C, it would be good to have a negative control with a mutated miR-310a.

-There is probably CLIP data available for FXR1. The authors should check if miR-310a is described as a binding target and determine the sequence that is recognized.

-Binding of PNPT1 to miR-301a is not shown.

-In fact, their model can have two components and they have to check that. It looks like that binding and function of FXR1 to p21 transcript is influenced by miR301 binding and vice versa. Are miR301a and FXR1 binding sites in p21 3'UTR close by? They can check for instance using luciferase constructs if mutation of miR301a binding site in p21 3'UTR abolishes FXR1 binding and regulation. Similarly, they can mutate FXR1 site and determine if miR301a regulation is affected.

Minor points:

-The authors should check the expression correlation between FXR1 and miR-301a in TCGA head and neck cancer and impact on survival.

-The introduction is somehow confusing and goes back and forth talking about miRNAs and RBPs.

-The authors should try to go beyond their model. This could really increase the value of their article. miRNAs and RBPs can be part of regulatory networks, for instance Musashi1 and miR-137 (31004009). Using available data and predictions, they could check if FXR1 and mir301a co-regulate other target genes.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: No

Reviewer #3: No

Dear Dr Palanisamy,

We are pleased to inform you that your manuscript entitled "RNA Binding Protein FXR1-miR301a-3p axis contributes to p21WAF1 degradation in oral cancer" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional accept, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about one way to make your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Michael V. Autieri

Guest Editor

PLOS Genetics

David Kwiatkowski

Section Editor: Cancer Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #2: I am satisfied by the revisions both in the results and discussion sections.

Reviewer #3: Authors successfully addressed my questions and I think the paper is ready for publication.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #2: Yes

Reviewer #3: None

**********

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: No

Reviewer #3: No

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-19-01404R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.