PONE-D-25-50225Labeling of Nascent RNA in the C. elegans IntestinePLOS ONE

Dear Dr. Weber,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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3. Does the protocol describe a validated method?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

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Reviewer #1: This paper reports a new method for detecting nascent ribosomal RNA (rRNA) in C. elegans intestinal cells. To overcome the impermeability of the C. elegans cuticle, the intestine is removed by dissection for incubation with EU, a uridine derivative that can be labeled by click chemistry for quantitative light microscopy. The method is clearly described and accompanied by a detailed protocol posted at protocols.io. Quantitative imaging detects a robust EU signal in the nucleolus, the established site or rRNA synthesis. RNAi of a canonical transcription factor for rRNA expression depletes the nucleolar EU signal lending strong support to the hypothesis that this method reliably detects nascent rRNA. Lastly, live cell markers (GFP) for nucleolar compartments appear to confirm that rRNA is largely localized to the fibrillar zone (FZ) (see below). This manuscript offers a useful and well-validated protocol for measuring rRNA in the C. elegans intestine and could be potentially useful for detection of nascent RNA in other C. elegans dissected tissues or isolated cells.

Minor Revisions:

1. A key finding is that rRNA transcripts are localized to the nucleolar fibrillar zone (FZ). Figure 3B quantifies co-localization (Spearman correlation coefficient) of the EU-594 signal with FZ markers DOA-1 and GARR-1 vs the granular zone (GZ) marker NUCL-1. Student’s t-test shows no significant difference (ns) between DOA-1 and GARR-1 but no statistics are reported for a comparison of NUCL-1 vs DOA-1 and GARR-1. This statistical test is important for substantiating the authors’ conclusion that the “majority of nascent rRNA transcripts localize to the fibrillar zone.“

2. Because intestinal cell nuclei are polyploid, it should be possible to detect other classes of nascent transcripts. This finding would provide a useful demonstration of the broad applicability of the protocol.

3. Need reference for concentration of ActD sufficient to inhibit RNA Pol II (line 154)

4. A figure illustrating click chemistry would be useful to readers unfamiliar with this method.

Reviewer #2: In this manuscript, Gholamalamdari and Weber present a protocol for nascent labelling of RNA in dissected intestines in C. elegans. Historically, the worm cuticle has prevented facile labelling of RNA with nucleoside analogs. Here, the authors solve this problem by extrusion of the intestine, allowing efficient 5-ethynyl uridine (EU) incorporation. The authors provide good evidence for nascent transcript labelling in the nucleolus, demonstrating reduced incorporation in response to perturbations of transcription, such as treatment with actinomycin D or knockdown of tif-1a (RNA polymerase I transcription factor). Subsequently, the authors further use this technique to explore the location of rRNA transcription within the nucleolus, by co-staining nascent RNA with known markers of the Fibrillar Zone (FZ) and Granular Zone (GZ). Their results demonstrate that the EU signal localizes within the FZ.

Overall, I enjoyed reading this protocol and I think it will be a good addition for the C. elegans community interested in studying gene regulation and transcription.

I only have minor comments that would be helpful to aid others interested in trying this technique.

1) It would be good for the authors to provide some rough guidelines/recommendations on how many animals should be dissected for a typical imaging experiment. It’s not clear how much material loss takes place after the manipulations.

2) Do the authors have some lower magnification images of the labelled intestines? Does the nucleolar/rRNA signal make it difficult to observe mRNA in the nucleus or cytoplasm? This would be interesting to know for anyone interested in studying other RNA species in the cell.

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Reviewer #1: No

Reviewer #2: No

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<p>Labeling of nascent RNA in the C. elegans intestine

PONE-D-25-50225R1

Dear Dr. Weber,

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,

Eric Jan, Ph.D.

Academic Editor

PLOS One

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