Metabolic and circadian inputs encode anticipatory biogenesis of hepatic fed microRNAs

Unravelling molecular and physiological mechanisms that couple circadian and metabolic cues to regulate anticipatory miR biogenesis during fed-fast-refed cycles in the liver.


A. THESE ITEMS ARE REQUIRED FOR REVISIONS
--A letter addressing the reviewers' comments point by point.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max.200 characters including spaces).This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title.It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person.Author names should not be mentioned.
--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-feeB. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): In the submitted manuscript titled 'Metabolic and Circadian Inputs Encode Anticipatory Biogenesis of Hepatic Fed MicroRNAs' by Sandra et al, the authors have followed the initial finding reported earlier by the same group (Maniyadath et al., 2019) to identify the oscillatory expression of certain miRNAs that are known to be controller of important starvation responsive genes.The investigation reported here, they have concluded an interesting hypothesis explaining the observation.They have scored the anticipatory high expression of pri-and pre-miRNA of corresponding mature miRNAs that are shown to be down regulated in the process of starvation.This hypothesis has been tested in the context of day light cycle and circadian rhythm and in the context of hypoglycemic and hyperglycemic conditions induced by pharmacological agents.I appreciate the approaches the authors have taken but I am disappointed of not finding any "real" mechanistic insight at molecular level that can explain these observations.The key concerns I have: 1.How the author can be certain that the upregulation (anticipatory) of pr-and pre-miRNAs are happening at post-transcription level and not due to a processing defect caused by the lowering of some components of miRNA machineries that are known to be regulated (like Dicer, or DGCR8) under different metabolic context.The expression of these components should be checked.2. To confirm the regulation happening at transcriptional level, the authors must have done experiments by doing DNA CHIP assay to confirm the enhanced association of the polymerase with the promoter region of the responsive miRNA expressing locus in starvation .It is also important to have control with the miRNA gene locus that are not expressing in the same manner during starvation-fed cycle.3. The extracellular export of miRNA can also contribute to the level of changed miRNA.It will be wise to look at the circulatory level of target miRNAs, pre-and pri-miRNAs levels in the serum of the animals going through fed and starvation cycle.4. The anticipatory differential expression of mature and precursor levels of miRNAs at the global level should be wisely followed in an bioinformatic approach involving gene expression data from normal fed and starved animal liver.This is essential to find a true link related to metabolic dysregulation and conciliatory miRNA expression-the basis of the hypothesis.
Reviewer #2 (Comments to the Authors (Required)): Anticipatory behavior and physiological response to food is particularly important for animals to restore metabolic homeostasis promptly and increase the survival/fitness.miRNAs have been identified as important posttranscriptional regulators of gene expressions including hepatic metabolism.In this manuscript, the authors report several hepatic miRNAs that respond to fed at the biogenesis level.Overall this is an interesting paper mainly focusing on the biogenesis of miRNAs responding to metabolic stages.The findings are interesting and the current evidence seem solid.However, I do have a few things that need to be addressed.1.In the abstract, the authors mentioned that "we report anticipatory biogenesis of oscillatory hepatic microRNAs that peak during a fed state and inhibit starvation responsive genes".This might have been reported by the authors and others in previous papers, however, I don't see any "inhibit starvation responsive gene"expression data throughout the current manuscript.Indeed, this is also one of the major caveats for this manuscript.The authors systematically checked the biogenesis of the four miRNAs under different conditions, however there is almost no data of their effects on potential targets.I suggest the authors used their samples to run a few qPCR or even WB to check several potential miRNA targets that might be starvation related here.
1st Authors' Response to Reviewers January 14, 2024 In the submitted manuscript titled 'Metabolic and Circadian Inputs Encode Anticipatory Biogenesis of Hepatic Fed MicroRNAs' by Sandra et al, the authors have followed the initial finding reported earlier by the same group (Maniyadath et al., 2019) to identify the oscillatory expression of certain miRNAs that are known to be controller of important starvation responsive genes.The investigation reported here, they have concluded an interesting hypothesis explaining the observation.They have scored the anticipatory high expression of pri and pre-miRNA of corresponding mature miRNAs that are shown to be down regulated in the process of starvation.This hypothesis has been tested in the context of day light cycle and circadian rhythm and in the context of hypoglycemic and hyperglycemic conditions induced by pharmacological agents.I appreciate the approaches the authors have taken but I am disappointed of not finding any "real" mechanistic insight at molecular level that can explain these observations.The key concerns I have: 1. How the author can be certain that the upregulation (anticipatory) of pr-and pre-miRNAs are happening at post-transcription level and not due to a processing defect caused by the lowering of some components of miRNA machineries that are known to be regulated (like Dicer, or DGCR8) under different metabolic context.The expression of these components should be checked.

Response:
We thank the reviewer for this critical input.We have addressed this concern by quantifying the expression of DROSHA and DICER proteins in fed-fast-refed paradigms as suggested by the reviewer.These results have been incorporated in the revised manuscript (Fig S4B-C).To elaborate, while the levels of DROSHA did not alter, we observed a starvation-dependent decrease and a refed-associated increase in the levels of DICER.The elevated DICER levels in the refed state correlate well with the upregulation of mature miRs during refeeding and posit a metabolic control of microRNA maturation via DICER.2. To confirm the regulation happening at transcriptional level, the authors must have done experiments by doing DNA CHIP assay to confirm the enhanced association of the polymerase with the promoter region of the responsive miRNA expressing locus in starvation.It is also 1.At the outset we would like to point out that in our published work (Maniyadath et al., 2019), we have exhaustively characterized the target mRNAs of these miRs and how they correlate with mRNA and protein expression of pathways relevant to fed-fast-refed cycles.In addition to extensive bioinformatic analysis, this study also provided a mechanistic basis for miRmediated downregulation of genes that lead to metabolic deficits.

4B-C and Fig
2. The focus of the current manuscript was to unravel the anticipatory biogenesis of the hepaticfed microRNAs and to uncover the upstream regulators which is a follow-up of the previous study that contains the kind of analysis the referee is pointing towards.
3. Furthermore, in the current manuscript, we have checked the levels of the prominent target genes (starvation transcripts) in ad libitum fed, light-fed, and dark-fed groups (Fig S1C and Anticipatory behavior and physiological response to food is particularly important for animals to restore metabolic homeostasis promptly and increase the survival/fitness.miRNAs have been identified as important posttranscriptional regulators of gene expressions including hepatic metabolism.In this manuscript, the authors report several hepatic miRNAs that respond to fed at the biogenesis level.
Overall this is an interesting paper mainly focusing on the biogenesis of miRNAs responding to metabolic stages.The findings are interesting and the current evidence seem solid.
We thank the reviewer for the positive comments and we are encouraged that the results presented in the manuscript were found to be of interest.
However, I do have a few things that need to be addressed.
1.In the abstract, the authors mentioned that "we report anticipatory biogenesis of oscillatory hepatic microRNAs that peak during a fed state and inhibit starvation responsive genes".This might have been reported by the authors and others in previous papers, however, I don't see any "inhibit starvation responsive gene"expression data throughout the current manuscript.Indeed, this is also one of the major caveats for this manuscript.The authors systematically checked the biogenesis of the four miRNAs under different conditions, however there is almost no data of their effects on potential targets.I suggest the authors used their samples to run a few qPCR or even WB to check several potential miRNA targets that might be starvation related here.

Response:
We respectfully submit that the reviewer has possibly missed the qPCRs done for target mRNAs which were included in the original submission.Nonetheless, the data presented in Fig SIC and Fig 2D provide an exhaustive characterization of target mRNA expression which is consistent with our published work (Maniyadath et al., 2019) and clearly demonstrate an anticorrelated pattern with respect to the mature miRs.
2. This manuscript lacks explanations of the rationale to study these four oscillatory miRNAs in the beginning of their results.Is this from a screen?Why do they start from these four?If just because they identified these four miRNAs in the previous study, then why they want to do it again?What new does this study provide?This needs to be briefly addressed somewhere in the manuscript.

Response:
We agree with the reviewer that the original version of the manuscript lacked a clear explanation in this regard.We have now addressed this concern in the revised manuscript at the beginning of the results section.Thank you for submitting your revised manuscript entitled "Metabolic and Circadian Inputs Encode Anticipatory Biogenesis of Hepatic Fed MicroRNAs".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Along with points mentioned below, please tend to the following: -please be sure that the authorship listing and order is correct -please add ORCID ID for the corresponding author --you should have received instructions on how to do so -it looks like there is a discrepancy in the presentation of the name of one of your co-authors: U.S. Sandra in the manuscript file vs. Sandra US in the system-please correct -the full name (middle name as initials) of each author should be given on the title page -there is only one panel in Figure S2, so there is no need to label it as A. Please correct the figure and its legend and call out in the manuscript text accordingly -we encourage you to revise the figure legend for Figure 5 such that the figure panels are introduced in an alphabetical order If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
LSA now encourages authors to provide a 30-60 second video where the study is briefly explained.We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608).Corresponding or first-authors are welcome to submit the video.Please submit only one video per manuscript.The video can be emailed to contact@life-science-alliance.orgTo upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.

A. FINAL FILES:
These items are required for acceptance.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max.200 characters including spaces).This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title.It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person.Author names should not be mentioned.

B. MANUSCRIPT ORGANIZATION AND FORMATTING:
Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.**Submission of a paper that does not conform to Life Science Alliance guidelines will delay the acceptance of your manuscript.****It is Life Science Alliance policy that if requested, original data images must be made available to the editors.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original data images prior to final submission.****The license to publish form must be signed before your manuscript can be sent to production.A link to the electronic license to publish form will be available to the corresponding author only.Please take a moment to check your funder requirements.****Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript.If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.**Thank you for your attention to these final processing requirements.Please revise and format the manuscript and upload materials within 7 days.
The authors have critically and experimentally addressed all my concerns and I recommend acceptance of this revised manuscript for publication in LSA.Congratulations to the authors.The final published version of your manuscript will be deposited by us to PubMed Central upon online publication.Your manuscript will now progress through copyediting and proofing.It is journal policy that authors provide original data upon request.
Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript.If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.***IMPORTANT: If you will be unreachable at any time, please provide us with the email address of an alternate author.Failure to respond to routine queries may lead to unavoidable delays in publication.***Scheduling details will be available from our production department.You will receive proofs shortly before the publication date.Only essential corrections can be made at the proof stage so if there are any minor final changes you wish to make to the manuscript, please let the journal office know now.

DISTRIBUTION OF MATERIALS:
Authors are required to distribute freely any materials used in experiments published in Life Science Alliance.Authors are encouraged to deposit materials used in their studies to the appropriate repositories for distribution to researchers.
You can contact the journal office with any questions, contact@life-science-alliance.orgAgain, congratulations on a very nice paper.I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially.We look forward to future exciting submissions from your lab.Sincerely, Eric Sawey, PhD Executive Editor Life Science Alliance http://www.lsajournal.org

Fig
Fig 2D) and observed an anti-correlated pattern with respect to the mature miRs consistent with our earlier publication.

February
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.
Thank you for submitting your Research Article entitled "Metabolic and Circadian Inputs Encode Anticipatory Biogenesis of Hepatic Fed MicroRNAs".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.