Dear Yehu,

Thank you for submitting your revised manuscript entitled "Characterization of RNA interference in the model cnidarian Nematostella vectensis reveals partial target silencing but lack of small RNA amplification" for consideration as a Short Report by PLOS Biology. Please accept my apologies for the delay in getting back to you as we consulted with an academic editor about the revised version.

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Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

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Dear Yehu,

Thank you for your patience while we considered your revised manuscript "Characterization of RNA interference in the model cnidarian Nematostella vectensis reveals partial target silencing but lack of small RNA amplification" for publication as a Short Report at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

Based on the reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers. Reviewer's #2 and #3 both note that the conclusions regarding siRNA-mediated antiviral responses should be toned down given the additional experiments do not demonstrate this definitively. In addition, Reviewer #2 raises some concerns with the documentation and controls for the experiment using self-replicating RNA. After discussions with the Academic Editor, while we agree that this additional work would be helpful, we ask that the limitations of the experiment are clearly stated and caveated in the manuscript text.

In addition, please also make sure to address the following editorial and data-related requests that I have provided below (A-G):

(A) We would suggest a very minor edit to the title, as follows, to help with flow. Please ensure you change both the manuscript file and the online submission system, as they need to match for final acceptance:

"Characterization of RNA interference in the cnidarian Nematostella vectensis reveals partial target silencing but lack of small RNA amplification"

(B) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

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- If you are reporting experiments where n ≤ 5, please plot each individual data point.

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As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

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Best wishes,

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

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Reviewer remarks:

Reviewer #1: The authors have provided clear and comprehensive responses to all my comments and questions, which has strengthened the manuscript. I fully support its publication.

Reviewer #2: Admoni et al. have analyzed features of RNA interference in the cnidarian model Nematostella vectensis. dsRNA injection into zygotes was used to induce RNAi and analyze its efficiency and other features. Authors show that Nematostella has a simple RNAi pathway without siRNA amplification and propose that RNAi in Nematostella provides only a short term antiviral mechanism.

The results are important for understanding evolution of dsRNA response and RNAi in particular as it is closely linked to the evolution of innate antiviral immunity. The revised version of the manuscript has addressed most points raised previously and I appreciate authors' efforts to address features of small RNAs generated from injected long dsRNA. I have two major issues with the revised version, which should be addressed before publication. In the minimal version both concern text revision.

Major issues

1) Authors should be precise and avoid overinterpreting their results. It has been pointed out during the first review and the issue still persists while I find it unnecessary. Authors examine dsRNA response and demonstrate presence of the canonical RNAi pathway = sequence-specific mRNA degradation induced by long dsRNA. The work on dsRNA response in this model is significant and sufficient. The fact that viral infection cannot be interrogated directly cannot excuse the use of the term of "antiviral RNAi". It does a huge disservice to the paper. Authors should critically revise all instances, where testing dsRNA response is called antiviral immune response, antiviral RNAi, antiviral response.

2) What is evidence that FHV-eGFP mRNA is actually a self-replicating system generating dsRNA? This experiment (Fig. 4A-F) is the most questionable in the manuscript - 1) a proper control would not be mCherry mRNA but FHV-eGFP mRNA with an RdRP-inactivating mutation, 2) there is no evidence of antisense strand synthesis, dsRNA presence, siRNA etc. This experiment does not bring any positive contribution to the manuscript (In my view, it does the opposite). It should be either removed or properly overhauled. A self-replicating system would strengthen the work only if it would be well documented.

Minor issues

1) Abstract 1st sentence "RNA interference (RNAi) is a sequence-specific mRNA degradation mechanism, which serves as an antiviral pathway by processing viral double-stranded RNA (dsRNA) into short interfering RNAs (siRNAs), leading to virus destruction. Please, revise as it would be confusing to general audience - it should state that siRNAs are guides for the sequence-specific mRNA degradation and indicate that antiviral role is one of roles of RNAi.

2) Abstract and elsewhere - please, clearly distinguish between innate immunity from acquired immunity, otherwise a text like "(anemones) possess an immune response with more vertebrate characteristics than previously thought" becomes misleading.

3) Figure 2 legend in the text on page 8 starts with the panel G while Fig. 2 has panels A-D - perhaps some automatic numbering ruined that.

4) Please, include more detailed description of spikeins and normalization in the methods - at the moment, normalization to million of reads is more comprehensible/informative about abundance of small RNAs than the relative numbers from spikein normalization in main figures.

5) I disagree with authors interpretation that "The highly reproducible pattern of the mapping suggests that Dicer has a sequence preference for processing of the injected dsRNA, as seen in previous experiments in mammalian cells." Such a pattern is likely highly influenced by strand selection (mentioned in the next sentence) and thus might also arise if the processing would be entirely random. Since authors do not observe strong phasing, it is plausible that the reproducible mapping pattern largely reflects strand selection of stochastically processed dsRNA.

6) Is Dicer in Nematostella processive? i.e. uses ATP-dependent helicase domain to process dsRNA - for an innate immunity RNAi system one would expect that it would be. Authors should discuss the helicase domain and true processivity in the discussion, as this feature is important for placing their results into the context of other analyses of Dicer and its ability to process long dsRNA.

Reviewer #3: The revised manuscript presents new data supporting a weak and transient siRNA response in Nematostella vectensis triggered by either injected dsRNA or self-replicating RNA. While I acknowledge the biological and technical constraints of this model system, I strongly recommend that the authors temper their conclusions and reframe key statements to describe a "likely antiviral siRNA response" rather than stating it as definitive (see lines 41-42 and 441-442).

Additionally, I have several minor comments:

L. 24: Please review the definition of RNAi in the abstract. RNA interference isn't just an antiviral pathway.

L. 90: Should be AGO1 instead of AGO.

L. 143 and other p values: show only the two first integers.

Supplementary Figure 1C-D are not mentioned in the text.

L. 293-294: in flies or mosquitoes reverse transcription of viral RNA into viral DNA is necessary for amplification of the siRNA response but it's not a prerequisite to produce antiviral siRNAs. I suggest adding ref. 17 here.

Dear Yehu,

On behalf of my colleagues and the Academic Editor, Rene Ketting, I am pleased to say that we can accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

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We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Best wishes, 

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

California (U.S.) corporation #C2354500, based in San Francisco