1. Author Response:

    Reviewer #2 (Public Review):

    Campylobacter jejuni is serious food-borne pathogen and understanding how the various products necessary for pathogenesis are regulated is a key step in preventing its growth and/or treating disease. Here, Sharma and coworkers demonstrate the complex pathway that leads to the maturation of two complementary regulatory RNAs and how one of the RNAs antagonizes the other to relieve repression of a virulence-related gene. The work is detailed and convincing, and provides a reference point for the roles of regulatory RNAs in C. jejuni as well as other bacteria. Future work will be needed to better understand when each of these RNAs is best expressed and processed into active form, and to fully support the idea that one RNA acts as an antagonist for the other.

    We thank the reviewer for their positive feedback on our work. Additional experiments (Figure 7B) provide additional evidence that CJnc180 is an antagonist of CJnc190 and affects ptmG.

    Reviewer #3 (Public Review):

    In this manuscript the authors describe the biogenesis and the mechanism of action of a pair of cis-encoded sRNAs: CJnc190 and CJnc180. Both RNAs are being processed by RNase III. 5' and 3' ends mapping together with in vitro and in vivo experiments using purified RNase III and rnc deletion mutant demonstrated that the processing of CJnc190 sRNA depended on the formation of an intramolecular duplex, while CJnc180 sRNA processing required the presence of the antisense CJnc190 sRNA. The mature CJnc190 and CJnc180 sRNA specious are 69 and 88 nt long respectively. They also show that mature CJnc190 sRNA represses translation of ptmG via base-pairing and CJnc180 sRNA antagonizes CJnc190 repression acting as a sponge, scavenging CJnc190 sRNA. In addition, they find that two promoters are responsible for the synthesis of CJnc190 sRNA and both transcripts are subject to RNase III processing.

    The study represents an enormous amount of work. The data are solid and generally support the overall conclusions. Having said that the manuscript is overwhelming, loaded with too many details which make the reading difficult and in the absence of a bigger picture many times uninspiring.

    We thank this reviewer for the overall positive feedback. We agree that this is a very complex story. We have made several revisions to the text and Figures and have moved details to the Supplementary Information. We hope this facilitates reading of our manuscript.

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  2. Evaluation Summary:

    Campylobacter jejuni is serious food-borne pathogen and understanding how the various products necessary for pathogenesis are regulated is a key step in preventing its growth and/or treating disease. Here, Sharma and coworkers describe the complex pathway that leads to the maturation of two complementary regulatory RNAs and how one of the RNAs antagonizes the other to relieve repression of a virulence-related gene.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the reviewers.)

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  3. Reviewer #1 (Public Review):

    The manuscript describes the mechanisms of biogenesis of two antisense sRNAs by RNase III in C. jejuni, CJnc180 and CJnc190, as well as the specific post-transcriptional activity of CJnc190 on ptmG. The study provides thorough experimental support of (i) binding of CJnC190 to repress translational of ptmG, (ii) RNAse III processing to produce mature CJnc190 and CJnc180 transcripts, (iii) location and contribution of CJnc180/190 promoters and 3' ends, and (iv) mechanisms of RNase III cleavage of CJnc180 and CJnc190. Notably, this study proposes a novel cis-sRNA processing mechanism of CJnc180 in which base pairing with antisense sRNA CJnc190 facilitates proper cleavage by RNase III. Overall, this well constructed and informative study provides impactful knowledge that furthers the field of regulatory RNAs.

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  4. Reviewer #2 (Public Review):

    Campylobacter jejuni is serious food-borne pathogen and understanding how the various products necessary for pathogenesis are regulated is a key step in preventing its growth and/or treating disease. Here, Sharma and coworkers demonstrate the complex pathway that leads to the maturation of two complementary regulatory RNAs and how one of the RNAs antagonizes the other to relieve repression of a virulence-related gene. The work is detailed and convincing, and provides a reference point for the roles of regulatory RNAs in C. jejuni as well as other bacteria. Future work will be needed to better understand when each of these RNAs is best expressed and processed into active form, and to fully support the idea that one RNA acts as an antagonist for the other.

    Read the original source
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  5. Reviewer #3 (Public Review):

    In this manuscript the authors describe the biogenesis and the mechanism of action of a pair of cis-encoded sRNAs: CJnc190 and CJnc180. Both RNAs are being processed by RNase III. 5' and 3' ends mapping together with in vitro and in vivo experiments using purified RNase III and rnc deletion mutant demonstrated that the processing of CJnc190 sRNA depended on the formation of an intramolecular duplex, while CJnc180 sRNA processing required the presence of the antisense CJnc190 sRNA. The mature CJnc190 and CJnc180 sRNA specious are 69 and 88 nt long respectively. They also show that mature CJnc190 sRNA represses translation of ptmG via base-pairing and CJnc180 sRNA antagonizes CJnc190 repression acting as a sponge, scavenging CJnc190 sRNA. In addition, they find that two promoters are responsible for the synthesis of CJnc190 sRNA and both transcripts are subject to RNase III processing.

    The study represents an enormous amount of work. The data are solid and generally support the overall conclusions. Having said that the manuscript is overwhelming, loaded with too many details which make the reading difficult and in the absence of a bigger picture many times uninspiring.

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  6. Evaluation Summary:

    Campylobacter jejuni is serious food-borne pathogen and understanding how the various products necessary for pathogenesis are regulated is a key step in preventing its growth and/or treating disease. Here, Sharma and coworkers describe the complex pathway that leads to the maturation of two complementary regulatory RNAs and how one of the RNAs antagonizes the other to relieve repression of a virulence-related gene.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    Read the original source
    Was this evaluation helpful?
  7. Reviewer #1 (Public Review):

    The manuscript describes the mechanisms of biogenesis of two antisense sRNAs by RNase III in C. jejuni, CJnc180 and CJnc190, as well as the specific post-transcriptional activity of CJnc190 on ptmG. The study provides thorough experimental support of (i) binding of CJnC190 to repress translational of ptmG, (ii) RNAse III processing to produce mature CJnc190 and CJnc180 transcripts, (iii) location and contribution of CJnc180/190 promoters and 3' ends, and (iv) mechanisms of RNase III cleavage of CJnc180 and CJnc190. Notably, this study proposes a novel cis-sRNA processing mechanism of CJnc180 in which base pairing with antisense sRNA CJnc190 facilitates proper cleavage by RNase III. Overall, this well constructed and informative study provides impactful knowledge that furthers the field of regulatory RNAs.

    Read the original source
    Was this evaluation helpful?
  8. Reviewer #2 (Public Review):

    Campylobacter jejuni is serious food-borne pathogen and understanding how the various products necessary for pathogenesis are regulated is a key step in preventing its growth and/or treating disease. Here, Sharma and coworkers demonstrate the complex pathway that leads to the maturation of two complementary regulatory RNAs and how one of the RNAs antagonizes the other to relieve repression of a virulence-related gene. The work is detailed and convincing, and provides a reference point for the roles of regulatory RNAs in C. jejuni as well as other bacteria. Future work will be needed to better understand when each of these RNAs is best expressed and processed into active form, and to fully support the idea that one RNA acts as an antagonist for the other.

    Read the original source
    Was this evaluation helpful?
  9. Reviewer #3 (Public Review):

    In this manuscript the authors describe the biogenesis and the mechanism of action of a pair of cis-encoded sRNAs: CJnc190 and CJnc180. Both RNAs are being processed by RNase III. 5' and 3' ends mapping together with in vitro and in vivo experiments using purified RNase III and rnc deletion mutant demonstrated that the processing of CJnc190 sRNA depended on the formation of an intramolecular duplex, while CJnc180 sRNA processing required the presence of the antisense CJnc190 sRNA. The mature CJnc190 and CJnc180 sRNA specious are 69 and 88 nt long respectively. They also show that mature CJnc190 sRNA represses translation of ptmG via base-pairing and CJnc180 sRNA antagonizes CJnc190 repression acting as a sponge, scavenging CJnc190 sRNA. In addition, they find that two promoters are responsible for the synthesis of CJnc190 sRNA and both transcripts are subject to RNase III processing.

    The study represents an enormous amount of work. The data are solid and generally support the overall conclusions. Having said that the manuscript is overwhelming, loaded with too many details which make the reading difficult and in the absence of a bigger picture many times uninspiring.

    Read the original source
    Was this evaluation helpful?