PONE-D-23-04780The exquisite specificity of human protein arginine methyltransferase 7 (PRMT7) toward Arg-X-Arg sitesPLOS ONE

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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: Partly

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: This manuscript explores the substrate specificity of recombinant full length PRMT7 produced from E. coli using recombinant full length human and X.laevis histones and synthetic short peptides. Enzyme assays are carried out using [3H]-AdoMet assays, using SDS-PAGE/fluorographs for full length histones and P81 phosphocellulose filter paper assay for the peptides. The authors conclude that the Arg-X-Arg consensus is needed for the substrates to be methylated efficiently, where X = Gly or Lys, but not Glu. The surrounding sequences around the Arg-X-Arg motif is important, where Lys before or after the retains the Km indicating those to likely interact with PRMT7. Increase in salt concentration affects both Km and Vmax.

While the study support and add to the insight into PRMT7 activity, it is concise and limited to the histone H2B and a set of KG peptides.

The current study is not sufficiently extensive enough to be able to claim that PRMT7 has ‘exquisite’ specificity for Arg-X-Arg sequence as noted in the title. Firstly, because the sequence variation tested is very limited, and the claim can only be made with respect to mono verses di arginine sequences (mono- and di-arginine containing KG peptides). Secondly, of the KG peptide, the RER peptide, which has the Arg-Glu-Arg motif, is just as poor a substrate as the mono R peptide. Thus, it is not all Arg-X-Arg motif that are substrates. Thirdly, the spacing between the two arginine residues have not been tested thoroughly, and only Arg-Arg and Arg-Lys-Ser-Arg sequences are tested within the X.laevis histone / peptide context (H. sapiens H2B vs X. laevis H2B peptide/histones, where there are three arginines in both but with different spacings).

It appears that RXRXR is the most favoured (from this publication, as well as author’s previous JBC 2013 288(52) 37019-37025)? The authors do not discuss how the knowledge gained in this paper may help with understanding / confirming the PRMT7 cellular substrates (those identified to date in cells e.g. Life 2021 11(8)768?). Also it is important to emphasise the limitations of using peptides.

Specific points:

- Likely due to the formatting but the Figure legends and main text difficult to distinguish.

- Fig 2: Labelling of lane is incorrect? The figures do not support the conclusion in text “Line 194-194: “To visualize methylation of H2B substrates by PRMT7, we obtained 1-day and 74-day [3H]-fluorograph exposures of all samples (Figure 2). Only human histone H2B appeared to be methylated after a 1-day exposure.” Should sample lane 3 be GST-HsPRMT7 + Hs H2B? The 4th lane is appears to be wrong too? Should lane 3 and lane 4 labelling be swapped around? This figure is very confusing. Also any reason that 74 day exposure is chosen?

- line 240: “Taken together, the data suggest that the K30R/R31K substitutions in the 29-RKRSR-33 target sequence affects PRMT7 methylation activity by affecting the turnover rate kcat, more so than the substrate binding affinity, km.” ) In Figure 4, only two peptide concentrations tested – no detail kinetic data provided, so cannot discuss about kcat, km based on this data

- Line 242 - Please note that Km is not binding affinity.

- Fig 4/ Fig 6 – Figures need labelling (LHS, RHS) and noted in the legend.

- Figure 6. Difficult to distinguish between the GST-HsPRMT7 only and Ac-KKGGRGRGGKKY-amide peptides. They are both black circle;

- What is the Km of SAM for PRMT7? Is co-substrate saturating conditions at 0.14uM SAM that the assays are being carried out? Also difficult to check the activity as the y-axis are given in cpm. When Michaelis Menten plots are provided for Hs H2B(23-37) (Fig8), are the initial rates calculated for different substrate concentrations at linear range? The activity on all the other figures under these conditions appears not to be in the linear range.

- The KG peptide nomenclature is confusing – in particular when they are noted in a sentence e.g. “Line 342 - From the experiments shown in Figure 7, we show that the RGR KG, KRGR KG, RKR KG, and RGRK KG peptides have very similar binding affinities ranging from 0.57 to 0.85 μM”. Would help the readers if different names than KG are used so there is no mistaking that the “KG” is not part of the sequence.

- Are the plots for Fig7 the same as for Fig8 but with narrower concentration range?

- Table 2. Please provide errors for the Km and Vmax values. Please add nomenclatures for the peptides.

- While the statistical analysis has been carried out, it is difficult to see if the data analysis is carried out for three technical replicates for a single experiment, or three independent experiments. If the former, then more robust data is needed to support the work.

Reviewer #2: A very detailed analysis of the substrate specificity of PRMT7

Page 14 Paragraph starting line 226 is confusing because the increase in activity observed is due to the increased length of time of the experiment (40 mins vs 3 h). However, the author makes it sound like the increase of histone from 10 to 50 microM is the reason. Then in line 232 the author states that decreased methylation was observed with 50 microM histone compared to 10 microM. As measurements were only taken every hour, then the activity could be considered increasing linearly over 0-3 h (not 0-1).

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Reviewer #1: No

Reviewer #2: No

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The exquisite specificity of human protein arginine methyltransferase 7 (PRMT7) toward Arg-X-Arg sites

PONE-D-23-04780R1

Dear Dr. Clarke,

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,

A Ganesan

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

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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: Yes

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

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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

**********

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: The authors have addressed all concerns of the reviewers, and the manuscript is clearer to follow for the readers.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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