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Dear Dr Suter,

Thank you very much for submitting your Research Article entitled 'α-Phenylalanyl tRNA synthetase attenuates Notch signaling by competing with Notch through its N-terminal domain' to PLOS Genetics.

The 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 a much-revised version. We cannot, of course, promise publication at that time.

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Gregory P. Copenhaver

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Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: Ho et al. aims to determine whether and how alpha-Phenylalanyl tRNA synthetase (α-PheRS) promotes cellular growth and proliferation in tissue progenitors and how it may contribute to tumour formation.

Ho et al. suggest that α-PheRS blocks Notch signalling by competing with the Notch intracellular domain (NICD) for factors that promote downstream Notch target expression. The authors provided a series of experiments in a variety of tissues (larval and adult midgut, larval brain, larval wing imaginal discs and adult wings) to show that α-PheRS overexpression resembles the loss of Notch in larval and adult midgut progenitors, larval neuroblasts and wing imaginal disc cells. These effects included progenitor cell and enteroendocrine cell (EE) expansion in the adult midgut, the loss of type 1 and 2 neuroblasts in the larval midgut, and ectopic vein formation in the adult wing. Furthermore, the authors showed similar effects by overexpressing a likely aminoacylation-dead α-PheRS (α-PheRSCys), suggesting that the effects are not translation-dependent. They also showed that α-PheRS and α-PheRSCys overexpression can inhibit the Notch-responsive reporter, NRE-GFP, in the adult midgut and larval wing disc. Lastly, the authors suggest that α-PheRS competes to bind factors that interact with the NICD, such as Su(H). They provide experiments to support this hypothesis by attempting to rescue the effects of α-PheRS overexpression by NCID expression. Additionally, they isolated an N-terminal region of α-PheRS that may compete with NICD. To do this, the authors generated a truncated N-terminal-only α-PheRS (α-S), which when overexpressed can produce the same effects in adult midguts and larval and adult wings as the full-length version. Using Western analysis, the authors showed that α-PheRS is normally found in larvae in a variety of forms (55, 40 and 25 KDa) and that different forms can be found in different larval tissues. Using mass spectroscopy, the authors identified regions in the smaller variant (25 KDa) that contains overlapping regions with α-S. Using protein sequence analysis, the authors further identified a conserved tryptophan in α-PheRS found in RAM domains of NICD that may aid in binding to other factors. Finally, the authors showed that the effects of α-PheRS overexpression in the adult midgut are lost by the expression of the W112A mutant version.

While the authors did show effects of α-PheRS overexpression and the importance of its N-terminal region in mediating these effects, the evidence that the authors provided for its role in antagonising Notch is not convincing. This is mainly because the effects of alpha-PheRS overexpression often do not or only somewhat resemble the loss of Notch signalling in these tissues. Since there in no biochemical evidence provided that α-PheRS competes for factors that interact with NICD nor that the W112A mutation abrogates its binding to these factors, strong evidence is needed to claim that α-PheRS overexpression affects Notch signalling. Based on their data in the larval and adult midgut and their previous work (Ho et al., 2020), it does seem that α-PheRS plays a role in proliferation in certain contexts.

Major concerns:

1. The effects of α-PheRS overexpression often do not or only partially resemble the loss of Notch signalling.

a) Adult midgut: the authors claim that the α-PheRS or α-PheRSCys overexpression resembles the loss of Notch signalling in adult midgut progenitors by showing that α-PheRS or α-PheRSCys overexpression results in the expansion of escargot (esg)+ cells and Prospero (Pros)+ EEs (Fig. 1E, 2). While the quantification does show a mild increase in EE cells upon α-PheRS overexpression, the images in Fig. 1 do not show an increase in EE cell clusters characteristic of the loss of Notch in adult midgut progenitors (See Ohlstein and Spradling, 2007, Patel et al., 2015). Loss of Notch in adult midguts results in the expansion of cells that resemble ISCs/progenitors and express high levels of ISC or progenitor (Delta, E-cadherin) and EE (Pros, Sc) markers (Maeda et al., 2008, Patel et al, 2015, Chen et al., 2018).

It’s possible that the esg+ and EE cell number increases due to accelerated ISC proliferation and tissue hyperplasia. In support, the α-PheRS overexpression results in a mild increase in mitotic cells and hyperplasia in the larval gut (Fig. 3F-G). The authors don’t, however, specifically test (e.g., by clonal analysis) whether PheRS overexpression affects cell number or cell size (growth) in the adult midgut.

Furthermore, in Fig.2E and H, it is unusual that the midgut is devoid of EEs after the NCID expression because the midgut takes several (3 or more) weeks to turnover (Jiang et al., 2009, Antonello et al., 2015). NICD expression in progenitors results in their rapid differentiation into ECs without affecting existing Pros+ EEs.

The NRE-GFP levels in the adult midgut were measured using a Western blot. It would be more convincing to see NRE-GFP levels in the adult gut as shown for the larval wing disc.

b) Larval midgut: It is not clear at which larval stage the midguts in Fig. 3A-D were examined (Jiang and Edgar, 2009). Although there seems to be a similar increase in AMP numbers after α-PheRS overexpression or N loss in AMPs, it is not clear that the effects of α-PheRS overexpression resemble Notch. For example, does the increase in AMP number after α-PheRS overexpression result in an increase in EE precursors or peripheral cell loss in pupal midguts as described in Takashima et al., 2001 and Mathur et al., 2010?

c) Adult wing: the authors showed that α-PheRS overexpression does not result in a notched-wing; however, it causes ectopic vein formation. This suggests perhaps a mild effect on Notch signalling, but the ectopic vein formation could also be from effects on other pathways.

d) Larval wing disc: the authors showed that α-PheRS overexpression in the posterior compartment results in a complete loss in NRE-GFP, suggesting a strong loss of Notch signalling. However, the α-PheRS overexpression does not cause a massive overgrowth (or duplication?) in the disc, which can be observed in the loss of Notch disc in Fig. 5F. This disc abnormality or overgrowth (or the lack of in discs overexpressing α-PheRS) are too not described in the text.

The larval and adult wing data provided are incongruent. The effects of α-PheRS on the adult wing (ectopic vein formation) and larval wing (no disc overgrowth) suggest a mild effect on Notch. In contrast, α-PheRS overexpression in discs causes the loss of NRE, suggesting a strong effect on Notch. The authors need to reconcile these differences.

Perhaps a further analysis of Dl expression in establishing D/V Notch signalling or an analysis of Notch targets Wg or cut will help clarify this more.

e) Type 1 and type 2 neuroblasts: In contrast to what the authors claim, Notch is not involved in type 1 neuroblast maintenance, but is involved in type 2 neuroblast maintenance (Haenfler et al., 2012, Xiao et al., 2012, Li et al., 2016). It is not clear how type 2 neuroblasts are lost from the central brain upon α-PheRS overexpression. Are they lost due to cell death or by differentiation or transformation to type 1? The authors also do not show any further molecular changes that occur due to loss of Notch in type 2 neuroblasts (e.g., erm expression, Li et al., 2016). Although the central brain area is shown, the effect of the loss of Notch and the effect of α-PheRSCys and NICD on type 1 neuroblasts should be shown in Fig. 4.

2) NICD experiments: the authors used NICD to rescue the effects of α-PheRS overexpression. In the adult fly midgut, the overexpression of NICD results in rapid progenitor differentiation into ECs (Ohlstein and Spradling, 2007; Korzelius et al., 2014). Thus, it is not surprising that its expression suppresses esg+ cell expansion or reduces the number of abnormal, partially-differentiated ECs upon α-PheRS overexpression.

3) W112A mutant: The authors showed that α-PheRS W112A mutant overexpression is unable to have the same effects of α-PheRS overexpression on tissue homeostasis, suggesting that this W is critical for ability of α-PheRS to compete with NICD. However, it is not clear if the W112A mutant form is stably expressed. It is also not clear whether there is high enough homology between α-PheRS and Notch RAM domains to be certain that a domain similar to RAM exists within α-PheRS.

Minor concerns:

1. The authors showed an enrichment of α-PheRS in what are likely larval and adult midgut progenitors (Figs. 1A,C). Using a marker (e.g., esg) to mark the progenitors will solidify the claim that PheRS is enriched in progenitors. Is α-PheRS enriched in other progenitors?

2. The experiments in Fig. 1E should be part of Fig. 2. Fig. 1 does nicely show a time series of the development of the effects of α-PheRS overexpression. Similar data can be found in Fig. 2 A-F for 5 days. As they showed similar effects at 5 days, perhaps the data in Fig. 1E could be placed into supplemental data or selectively combined with Fig. 2. The data also in 3H is relevant to Fig. 2 and not Fig. 3, which focuses on the effects of α-PheRS on larval progenitors.

3. The authors suggest that they are counting “differentiating EBs.” From the text, it seems that they mean ECs that continue to express esg+ cells. Perhaps a marker for ECs (e.g., Pdm1 or Myo1A) could be used together with esg to show that these abnormal ECs still express progenitor markers. This is often observed with midgut hyperplasia or dysplasia in aging midguts (Biteau and Jasper, 2008).

4. The y-axis in Fig. 2H would be clearer if labelled %EE/total cells per region. Similarly, it should be edited for Fig. 2I. Total cells should include all cells in this case and not only ECs. EEs make up roughly 10% of all midgut cells (Ohlstein and Spradling, 2007).

5. In Fig. 4B, the y-axis could be clearer. Perhaps “ratio central brain/optic lobe size”.

6. In the figures, insets from images (e.g., Fig. 3 A’-D’) or images of different specimens (Fig. 4A) should not be labelled with ‘ or ‘’. Using ‘ suggests that each image are different channels of the first image presented. These should be labelled as separate panels as in Fig. 1A-D.

7. Scale bars are missing from panels C-H. Scale bars can be found in some panels but are missing from many others.

8. The titles of subsections and figure legends do not accurately describe the findings and can be more accurate.

9. In Fig. 4, the authors should make it clear that GFP is being shown in each panel.

10. esg is recessive, and thus should be written esg, not Esg. See Fig. 3.

11. A discussion of which cancers show upregulated α-PheRS and whether Notch signalling is tumour suppressive in these tissues will help express the significance of the work better.

12. The manuscript needs careful editing for clarity and accuracy.

Reviewer #2: uploaded as an attachment

Reviewer #3: In this manuscript Ho et al. investigated the role of the alpha subunit of the cytoplasmatic Phenylalanyl tRNA synthetase (α-PheRS) as a novel general repressor of Notch signalling. They showed an inhibitory effect on Notch signalling in different larval and adult Drosophila tissues by similarities of α-PheRS overexpression to Notch-RNAi phenotypes that were rescued by simultaneous expression of the Notch intracellular domain. Besides, the authors proved that protein levels of a Notch responsive element reporter are reduced upon overexpression of α-PheRS. Thereby expression of an α-PheRS variant (α-S) encoding for the N-terminal 200 amino acids and lacking the catalytic domain was sufficient to induce phenotypes similar to Notch loss of function, indicating that the inhibitory effect on Notch signalling is independent of the catalytic function of α-PheRS. α-S also shows a weak sequence similarity to the Notch intracellular domain which includes a conserved key tryptophan residue which was shown to be essential for α-S to attenuate Notch signalling. Although the paper is well written and overall comprehensive, the figures and data presentation are of poor quality and need revision.

Major comments:

1) Line 145 and following: The authors state that α-PheRS levels are higher in progenitor cells compared to differentiated cells in Drosophila larval and adult midguts. For this statement they compared signal levels of antibody stainings against α-PheRS in diploid cells to polyploid cells, but without using any characterized markers for the different cell types. Enteroendocrine cells and their progenitors are also diploid whereas Enteroblasts already undergo endocycles prior to differentiation and are also polyploid (Edgar et al. 2014). The authors should characterize the different cell types by established markers to clearly distinguish them and discuss with the findings in the Edgar paper and this paper from the Dominguez lab (Antonello et al. 2015). Also, for defining the “differentiating EB” they should use Enterocyte markers like discs large 1 to proof that they are not fully differentiated Enterocytes by negative staining.

2) Line 182: Do the authors have data or in silico predictions which show or predict that α-PheRS directly binds “common downstream targets” of Notch signalling? If not, this speculative statement should better be mentioned in the discussion.

3) The presence of several RNAi stocks in the material and methods part suggests that also knockdown experiments for the α-PheRS have been performed. This raises several interesting questions:

I. Are they able to accelerate differentiation through RNAi?

II. Enteroblast specific function downstream of Notch input in adult midguts should be investigated using klumpfuss-Gal4 driver combined with tracing system (Korzelius et al. 2019, Reiff et al. 2019)

III. At least accelerated differentiation needs to be discussed as it was previously observed in e.g. (Korzelius et al. 2014, Korzelius et al. 2019, Reiff et al. 2019, Zipper et al. 2020). At least, these papers should be discussed with the data in the present manuscript.

4) Microscope images of the same tissue within one figure should be orientated in the same direction for better comprehensibility. Textboxes and corresponding images should be aligned properly. Diagrams should have precise and coherent axis titles.

5) Figure 3 E – E’’ and F: In E’’ it looks like there are more PH3+ cells compared to E’, but F shows the opposite. The quantifications in F should be normalized to the total cell number. G: is the “intestinal region” somehow defined? 200 cells cannot be the whole gut.

Minor comments:

1) “Drosophila” should be written italic throughout the manuscript

2) In the summary (line 39) the authors write about “moderately elevating α-PheRS levels” in adult midguts. What do they mean with “moderately elevating” in an experiment with Gal4/UAS driven overexpression of α-PheRS? Did they perform qPCRs or Western Blots to proof these moderately elevated levels of α-PheRS?

3) In Figure 1 B,D: axis titles of diagrams are too small. E: images can be subtitled with E-E’’’,F’-F’’’ and so on, this would make it easier to refer to the different genotypes shown. “esgts>YFP” is hard to read (green letters on grey background), correct throughout all figures.

4) In Figure 2 H: It should be written what was quantified “percentage of..” like in I.

5) Figure 4: A and B: Data for insc>NICD is missing.

6) Figure 6: G: y-axis title incomplete, “percentage of..” like in 4)

7) Figure 7: A: identity and similarity of aligned sequences and a legend should be added.

Bibliography:

Antonello ZA, Reiff T, Ballesta-Illan E, Dominguez M. 2015. Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch. Embo j 34:2025-2041.

Edgar BA, Zielke N, Gutierrez C. 2014. Endocycles: a recurrent evolutionary innovation for post-mitotic cell growth. Nature Reviews Molecular Cell Biology 15:197-210.

Korzelius J, et al. 2014. Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells. Embo j 33:2967-2982.

Korzelius J, Ronnen-Oron T, Baldauf M, Meier E, Sousa-Victor P, Jasper H. 2019. The WT1-like transcription factor Klumpfuss maintains lineage commitment in the intestine. bioRxiv:590885.

Reiff T, Antonello ZA, Ballesta-Illán E, Mira L, Sala S, Navarro M, Martinez LM, Dominguez M. 2019. Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila. Embo j 38:e101346.

Zipper L, Jassmann D, Burgmer S, Görlich B, Reiff T. 2020. Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARγ-homolog Eip75B in Drosophila. Elife 9.

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Reviewer #1: No: A spreadsheet with numerical data and statistics was not provided.

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Attachments

Attachment

Submitted filename: PGENETICS-D-21-01190_Review.docx

Dear Dr Suter,

Thank you very much for submitting your Research Article entitled 'α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript. While two reviewers are satisfied with the revision, one reviewer raises a number of concerns about the main conclusion of the manuscript on the phenotypic similarity of alpha-PheRS overexpression and Notch loss-of-function, while acknowledging that the alpha-PheRS can block the Notch reporter. Please respond to the critique of the reviewer and revise the manuscript accordingly. I will weight the arguments of the reviewer against your response and revision while making the final decision.

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Please let us know if you have any questions while making these revisions.

Yours sincerely,

Ville Hietakangas

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

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 revised manuscript, the authors claim that alpha-PheRS blocks Notch signalling by competing with NICD for factors that promote Notch target expression. They claim that the effects of alpha-PheRS overexpression resembles the loss of Notch signalling in larval and adult midgut progenitors, larval neuroblasts and wing imaginal discs. Other than showing alpha-PheRS can block the Notch reporter (NRE-GFP) in larval wing discs, the authors have not convincingly shown that that the other effects they describe in the larval and adult midgut, larval neuroblasts and the adult wing resemble the loss of Notch. It is important for the authors to show this particularly because there is no biochemical evidence provided that alpha-PheRS competes for factors that interact with the NICD nor evidence of effects on Notch target expression.

Adult midgut: The authors insist that the effects of alpha-PheRS overexpression resemble the loss of Notch in the adult midgut. This is based on an expansion of esg+ cells and a mild increase in Pros+ EE cells. It is important to note that hyperproliferation in the gut can increase number of esg+ cells (e.g., see Biteau et al., 2008). This will also mildly increase Pros+ cells as new Pros+ cells are added to an epithelium already containing Pros+ EEs. The loss of Notch in adult midguts results in an expansion of ISC- like cells that express high levels of Delta (ISC- specific marker), esg, E-Cadherin, Armadillo and Pros (EE marker) (Maeda et al., 2008; Patel et al., 2015; Chen et al., 2018). There are cheap, publicly available antibodies for many of these markers (e.g., Delta, E-Cadherin, Armadillo). These N- ISC-like progenitors are often highly-adherent to each other due to the high E-Cadherin levels. Furthermore, the loss of Notch in adult midgut progenitors results in the formation of clusters of Pros+ EE cells, which is not observed with alpha-PheRS overexpression.

The authors claim that they cannot examine MARCM clones in the midgut to analyze cell number due to cell fate changes. This is not true as MARCM clones can be used to assess cell number within midgut clones despite cell fate changes (see Salle et al., 2017; Lee et al., 2009).

NICD expression in adult midgut progenitors results in rapid differentiation into ECs without affecting existing Pros+ EEs. The authors claim that a loss of EEs upon NICD expression has been described before, but this is not true. The papers cited by the authors (Micchelli and Perrimon, 2006; Zhai et al., 2017) did not examine the effects on Pros+ cells after NICD expression in adult midgut progenitors.

The authors have also not addressed why they utilized Western blot analysis to assess NRE-GFP levels in the midgut. NRE-GFP can be observed in the midgut (see Wang et al., 2020). The authors stated that an internal control compartment is necessary for in vivo analysis, but an experimental midgut can be compared to an independent control midgut not overexpressing alpha-PheRS.

It is not clear whether the effects of alpha-PheRS overexpression in the larval midgut resemble those of the loss of Notch. The authors show an increase in esg+ cells and a mild increase in Pros+ cells. The authors provide us with a quantification of Pros+ cells in the larval midgut, but do not provide images of this expansion. However, studies that have examined Notch signalling in the larval midgut have not described an expansion of esg+ cells and Pros+ cells. These studies have rather shown that the loss of Notch in the larval midgut results in an increase in pupal endocrine cells within AMP islands and a loss of peripheral cell formation in the larval midguts (Takashima et al., 2011; Mathur et al., 2010). This is something that the authors have not examined.

The authors have shown that the loss of Notch in the larval wing results in a loss of NRE-GFP and a notched wing. In contrast, the alpha-PheRS overexpression, which results in a loss of NRE-GFP, does not cause a notched wing. These differences have still not been reconciled by the authors. The authors suggest that compensatory proliferation helps alpha-PheRS overexpressing wings to reach proper organ size, but the authors have not provided this data.

The authors claim that alpha-PheRS overexpression blocks Notch signalling in larval neuroblasts, which results in the loss of type 1 and type 2 neuroblasts. However, Notch is not required for in type 1 neuroblast maintenance (Haenfler et al., 2012), but it is required for type II neuroblast maintenance (Haenfler et al., 2012; Xiao et al., 2012; Li et al., 2016). To show that the loss of type 2 neuroblasts is due to inhibiting Notch, the authors need to show that they are lost by alpha-PheRS overexpression due to molecular changes that occur due to the loss of Notch signalling in type 2 neuroblasts (e.g. erm expression, Li et al., 2016). From the data provided, it’s not clear if their loss is due to affecting Notch or by affecting another process. Are these type 2 neuroblasts overexpressing alpha-PheRS lost due to cell death or by differentiation or by transformation to type 1 neuroblasts?

Lastly, their new staining for alpha-PheRS in Fig. 1 does not support their claim that alpha-PheRS is enriched in progenitors. It seems to be expressed in all the cell types and seems enriched in some Pros+ cells, but not all (see Fig. 1E”). Thus, the authors should reconsider their statement about alpha-PheRS being enriched in stem cells/progenitors.

Reviewer #2: The authors now have strengthened an antagonizing function of alpha-PheRS on the Notch signaling pathway by additional experiments. Additional control experiments were also performed. Unfortunately, despite several approaches, a direct interaction of alpha-PheRS with Su(H) at DNA could not be shown. However, the proposed mechanism was now more cautiously considered as "possible" in several places in the manuscript by the authors. The title of the manuscript has also been changed accordingly. I have no additional criticisms or objections.

Reviewer #3: In this revised version the authors addressed carefully the majority of my comments and suggestions. Last major concern is still the technical problem of the image quality commented on before. The authors state that this was improved, but the downloadable PDF is still having the problem of poor image resolution which leaves me unable (again) to see proper images and/or read Y-Axis (Fig.2+6). So, I cannot judge this, but this needs to be sorted out between the editor and authors before publishing. Overall, when these issues are fixed I recommend this manuscript for publication and congratulate the authors for this nice work.

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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

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Dear Dr Suter,

We are pleased to inform you that your manuscript entitled "α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain" 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 acceptance, 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 making 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,

Ville Hietakangas

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

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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-21-01190R2

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.

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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.