PONE-D-22-10327The Selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporationPLOS ONE

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Additional Editor Comments:

Please correct the following typos:

p. 7, the 2nd last line of the 1st section: "shwon" to "shown".

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: PONE-D-22-10327

The Selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation

The manuscript is very well written, the experiments are appropriate and effective. Results convincing, statistical analyses appropriate. Nice to see using both rat and human cell models used, and that extra bands were validated.

There are only very minor comments:

Suggest italicizing SELENOP to indicate the gene/nucleotide sequence and non-italicized when referring to the protein, as this would provide clarity. It appears to be used once on p. 6, but inconsistently so. Technically, only human gene would be SELENOP, whereas mouse and rat would be selenop.

Please change uM to µM (e.g., bottom of p. 8 and a couple of instances within the text on p. 9 and in Figure 5), and ug to µg (p. 16), and uL to µL (p17).

Please bracket ‘respectively’ by commas (p. 9), and change ‘media was’ to either ‘medium was’ or ‘media were’ (on p. 9 and also a couple of times in the methods section, and in legends for Figure 5 & 6)

Insert comma after i.e. (p. 10)

Be consistent in use of either UTRs or UTR’s. Both are used (see first few sentences of discussion) interchangeably. Similarly, spacing between 3’UTR and 3’ UTR should be consistent.

End of first paragraph of discussion: Here, we have found…… (comma missing)

P 15: 200 ng … were….

P 16: 15 µL…. were…

P, 18: E. coli should be italicized

p-values should always be with the zero preceding the period (e.g., p=0.037 in figure 5B, not p=.037)

The only actual questions concerns Figure S2 – can the authors please verify that the same images weren’t accidentally used for both (left and right) or that we are not misinterpreting the use of it? The images seem identical, down to the same artifacts in the phosphor imager bands that are slightly broken the same way.

Reviewer #2: This is a very interesting manuscript that deepens our knowledge of the regulatory frame of selenoprotein P (SELENOP) translation, an intriguingly unique mRNA that contains 2 SECIS elements that direct insertion of up to 10 selenocysteines in the polypeptide chain. The authors identified for the first time an RNA binding protein, PTBP1, that binds to an intermediate region between the 2 SECIS elements and regulates translation efficiency of this mRNA. The manuscript is clearly written, and the logical flow of findings is very straightforward to follow, with the adequate experiments needed and appropriate controls. It will add valuable information to the field of selenoprotein translation. Nevertheless, some minimal comments below would, if addressed, enrich further the manuscript quality prior to publication.

COMMENTS

Introduction:

• "All but one of the human selenoproteins contain a single Sec codon in their coding region" - There is a second UGA sometimes recognized as Sec in Deiodinase type 2 (see PMID: 11425850). Please rephrase.

Methods:

• Has the SELENOP antibody used in this manuscript been validated?

• Statistical Analysis used in experiments is embedded in figure legends. It will be best organized if shown as a subsection of the Methods.

Discussion:

• "For example, SELENOP mRNA levels have been reported to dramatically increase in response to exercise stress in mice fed a normal diet but not those fed a high fat diet [16]." Is PTBP1 regulated by exercise? A discussion on common potential regulators of PTBP1 should be incorporated to enrich the discussion.

• "(...) it is likely that the 14 functional RNP complex may form co-transcriptionally and coincident with RNA splicing." Has any of the proteins recognized by the screening related to RNA splicing that are below the threshold chosen for the selection of peptides? This could be an interesting point to discuss further.

• This manuscript showed that mutation in the preSECIS1 sequence reduces translational efficiency. PTBP1 could, via binding to an NMD partner, contribute also to nonsense-mediated decay of SELENOP mRNA according to Se conditions. This possibility would also enrich the discussion.

Figures:

• Legend of Fig. 2 has "D" when it should be "E" for the peptide counts data.

Reviewer #3: This study reports investigation of conserved sequences in the 3’UTR of selenoprotein P, using RNA affinity chromatography and mass spec. The overall premise is sound, and the experimental rationale is well thought out, with appropriate controls.

The authors report identification of PTBP1 as the major RNA binding protein specifically interacting with the inter-SECIS region. They further use CRISPR/Cas9 genome editing to assess functional role, and report regulation in response to oxidative stress and selenium concentration.

They further report that the ΔpreSECIS mutation caused a ~2-fold reduction in steady state mRNA levels with a concomitant increase in protein production. This intriguing finding highlights avenues for future investigation into the complex regulatory mechanisms required for expressing this critically important but translationally challenging and perplexing protein.

A minor point that should be corrected on p. 7, line 12 – mutating is misspelled in this sentence, “Figure 3C shows that mutatng individual patches…”

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Reviewer #2: No

Reviewer #3: No

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The Selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation

PONE-D-22-10327R1

Dear Dr. Copeland,

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,

Hodaka Fujii, M.D., Ph.D.

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

Reviewer #3: 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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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 #2: 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 #2: 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: Very interesting paper, and the revisions look good. Thank you for taking the time to verify the image source.

Reviewer #2: The Authors have addressed all comments appropriately. Without further issues to be raised, this manuscript seems acceptable to publication into PLOS ONE.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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