PONE-D-21-06328

The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid stress response to loss of La protein by fission yeast

PLOS ONE

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[This work was supported by the Intramural Research Program (HD000412-31PGD) of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. The funders had no role in study design, data collection, interpretation, or decision to submit for publication.]

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Reviewers' comments:

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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

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

Reviewer #1: No

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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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 the manuscript submitted by Cherkasova et al, the authors provide the evidence that the sla1+ gene of S. pombe, which encodes La protein, mediates general amino acid control (GAAC) response concomitant with nuclear surveillance mechanisms. First, transcriptome analysis reveals that genes upregulated in sla1D cells exhibit highly significant overlap with GAAC genes. Second, GAAC gene upregulation is suppressed by rrp6 deletion. Third, the authors isolated sla1D spontaneous revertant (SSR), which suppresses slow growth phenotype in NH4 + -media, and identified an F32V mutation of any1+ gene in the SSR mutants by whole genome sequencing. Furthermore, 3H-leucine uptake of SSR- any1-F32V cells in NH4 + -media is more robust than by sla1D cells.

The manuscript presents noble findings regarding a function of sla1+ gene, and is suitable for PLOS ONE. However, several important information is missing as described below, therefore, I cannot support publication of the manuscript in its present form.

Specific comments:

1. The authors showed that sla1D cells exhibited increased levels of gdh2+ and aca1+ mRNAs in EMM-NH4+ -media, which has already been shown by the authors’ previous study (in ref [50]). And then, they showed that additional rrp6D (Fig 2) and any1D (Fig 5) mutations reduced the upregulation. The levels of the increase of gdh2+ mRNA in sla1D cells is not comparable between the experiments in Fig 2 and Fig 5; it was detected at approx. x8 (Fig 2B, right) and x3.6 (Fig 5, lanes 3-4), respectively. It is also the case for aca1+ mRNA; it was detected at approx. x6 (Fig 2B, right) and x4.5 (Fig 5, lanes 3-4). Is there any difference in experimental conditions between these experiments? Is this just within the experimental variations? The authors need to perform these experiments at least three times and present the results with standard deviations and p-values.

2. Expression levels of rpl8+ mRNA in sla1D cells grown in EMM-NH4+ -media is drastically reduced in the experiment in Fig 2A (lanes 11 and 12), but not in Fig 3E (lanes 3 and 4) and Fig 5 (lanes 3 and 4). If the authors perform these experiments under the same conditions, the difference should not be appeared.

3. It seems that the authors isolate five SSRs (SSR1-5), whose growth phenotype was shown in Fig 3C. SSR1 also shows robust suppression phenotype in addition to SSR4, SSR5 and SSR6. Why do they abandon further analysis for SSR1 including whole genome sequencing? The authors should describe the precise clone number of isolated SSR and the reason why they pick SSR4, SSR5 and SSR6 in the manuscript.

4. SSRs were indicated like “SSR-4b” but not “SSR4” in Fig 3C. What is the difference between SSRX and SSR-Xb? The authors should describe the difference in the figure legend or main text.

5. In Fig 5, the authors claimed that any1-F32V led to lower aca1+ mRNA levels than any1+ in the sla1D background (compare lanes 15-16 and 17-18, x3.6 and x2.1, respectively). However, the aca1+ mRNA levels of sla1D any1D double mutant that is integrated sla1+ is also lower than that of sla1D (compare lanes 3-4 and 15-16, x4.5 and 3.6, respectively), which should be comparable, considering the authors claim as described in lines 405-407. The result is not supportive. To confirm the claim, the authors need to present the results with standard deviations and p-values as described above and reevaluate the result.

6. Point mutation sometimes effects on the stability of the protein. The authors need to confirm any1+ and any1-F32V protein levels, in addition to the mRNA expression levels by northern blotting shown in Fig 5.

7. The authors conclude that sla1D mutant exhibited decreased leucine uptake in EMM-NH4+ media compared to WT, in addition, any1D mutant, sla1D any1D double mutant, and SSR5 exhibited greater uptake than WT (lines 412-414). To confirm these differences, again, the authors need to present the results with standard deviations and p-values.

Reviewer #2: The manuscript entitled "The leucine-NH4+ uptake regulator Any1 limits ..." by Cherkasova et al. first analyzed gene expression profiles of a sla1∆ mutant, lacking a tRNA processing factor La homologue in S. pombe, grown in three different N-source conditions and found that the mutant exhibits significant similarity to the wild-type cells under GAAC in transcriptome. In addition, the sla1∆ cells in NH4+ media showed up-regulation of a set of genes that are a part of the CESR-induced genes. The former regulation seems to be driven by nuclear tRNA surveillance since nuclear exosome inactivation by rrp6∆ dampens the up-regulation of the GAAC-related genes in the sla1∆ background. The authors stepped forward to isolate spontaneous suppressor mutants and found that a F32V mutation on the any1+ gene, encoding an arrestin-homologue, suppressed poor growth of the sla1∆ mutant on the NH4+ plate and supported growth of the surrounding any1+ cells. The any1-F32V sla1∆ cells incorporated Leu more efficiently even in the presence of NH4+ than the sal1∆ cells. The authors concluded that the tRNA processing factor La is involved in the GAAC response and Any1 specifically acts to support this signal transduction.

All the experiments seem to be performed technically rigorous and fit to the scientific standard of this field. Essentially, the data presented as figures and tables support the authors' notions. For example, they precisely described discrepancy between rrp6∆ effects on GAAC gene expression and those on growth in NH4+ media, which led to identification of a specific allele of any1+ as a suppressor of sla1∆ with a unique features. They appropriately cited previous paper especially in the transcriptome analyses of the sla1∆ cells. Thus, the reviewer essentially supports the publication of the manuscript in the journal of PLOS One. Before publication, the following minor points should be amended:

1) p. 23, line 581, p. 24, line 584; ug should be µg (micro gram).

2) p. 24, lines 583–584; it says that the Hybridization solution contained 100 µg/ml yeast RNA. Was the RNA prepared from budding yeast but not from fission yeast? Or, it might be "tRNA" but not "RNA."

3) p. 24, line 585; the company name should be "Fuji Film."

4) The gene names and boxes in Tables 1–7 are color-coded. However, there is no explanation of the colors.

5) It is reader-friendly if the standard gene name of every gene listed in the lower section of Tables 2–7 is indicated. In addition, the reviewer is skeptical that the current style of the Tables 1–7 is appropriate for publication even on line. They should be adequately reshaped for publication.

6) In Fig. 2B, it is difficult to recognize bar identity from pattern examples in its inset because the pattern examples were enlarged too much.

7) In Fig. 6, the unit of the axis should be "pmol/10^7 cells."

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Reviewer #1: No

Reviewer #2: Yes: Tohru Yoshihisa

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PONE-D-21-06328R1

The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast

PLOS ONE

Dear Dr. Maraia,

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.

==============================

Two experts in the field have now reviewed your manuscript.

As you will see in their comments, both reviewers highly evaluated your revised manuscript. However, Reviewer 1 still raises concerns about your manuscript from a statistical viewpoint, which might require reconsideration of your conclusion of the manuscript.

==============================

Please submit your revised manuscript by Jul 08 2021 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.

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• 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: http://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,

Reiko Sugiura, M.D., PhD.

Academic Editor

PLOS ONE

Journal Requirements:

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.

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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: (No Response)

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

**********

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

Reviewer #1: No

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 #1: Yes

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 #1: Yes

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 #1: Cherkasova et al. have provided convincing response to issues raised in the previous round of review. I will support its publication after addressing the point as described below.

The authors described in lines 437-440 that “Although these quantifications do not carry strong statistical significance, visual inspection of the northern blot internal controls provide additional evidence that the any1-F32V allele is more effective at suppressing aca1+ levels than is the any1+ allele.” Generally, “visual inspection” sometimes misleads conclusions. I strongly recommend the authors evaluate the result using statistical parameters, such as standard deviations, p values, and t-tests. If the authors cannot find statistical significance of aca1+ mRNA levels between lanes 15-16 and 17-18, they should reconsider the conclusion; “The data suggest that any1-F32V is partially effective as its integration suppresses aca1+ levels to greater extent than any1+.” in lines 444-445.

Reviewer #2: (No Response)

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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.

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Reviewer #1: No

Reviewer #2: Yes: Tohru Yoshihisa

[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.

The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast

PONE-D-21-06328R2

Dear Dr. Maraia,

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