Diurnal control of iron responsive element containing mRNAs through iron regulatory proteins IRP1 and IRP2 is mediated by feeding rhythms
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Review Commons)
Abstract
Background
Cellular iron homeostasis is regulated by iron regulatory proteins (IRP1 and IRP2) that sense iron levels (and other metabolic cues) and modulate mRNA translation or stability via interaction with iron regulatory elements (IREs). IRP2 is viewed as the primary regulator in liver, yet our previous datasets showing diurnal rhythms for certain IRE-containing mRNAs suggest a nuanced temporal control mechanism. The purpose of this study is to gain insights into the daily regulatory dynamics across IRE-bearing mRNAs, specific IRP involvement, and underlying systemic and cellular rhythmicity cues in mouse liver.
Results
We uncover high-amplitude diurnal oscillations in the regulation of key IRE containing transcripts in liver, compatible with maximal IRP activity at the onset of the dark phase. Although IRP2 protein levels also exhibit some diurnal variations and peak at the light-dark transition, ribosome profiling in IRP2-deficient mice reveals that maximal repression of target mRNAs at this time-point still occurs. We further find that diurnal regulation of IRE-containing mRNAs can continue in the absence of a functional circadian clock as long as feeding is rhythmic.
Conclusions
Our findings suggest temporally controlled redundancy in IRP activities, with IRP2 mediating regulation of IRE-containing transcripts in the light phase and redundancy, conceivably with IRP1, at dark onset. Moreover, we highlight the significance of feeding-associated signals in driving rhythmicity. Our work highlights the dynamic nature and regulatory complexity in a metabolic pathway that had previously been considered well-understood.
Article activity feed
-
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Review Commons - Revision Plan
Manuscript number: RC-2023-02228
Corresponding author(s): Gatfield, David
1. General Statements
We are grateful to the three Reviewers for their detailed assessment of our manuscript and are delighted about their very constructive and positive evaluations, highlighting the study’s novelty and rigor.
Briefly, the main points raised by Reviewers 1 and 3 do not involve additional experiments and are mostly about rethinking manuscript structure (e.g. moving data/analyses to the supplement or removing them altogether, as they distract from the main thrust of the story) and making the text overall less dense and more readable.
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Review Commons - Revision Plan
Manuscript number: RC-2023-02228
Corresponding author(s): Gatfield, David
1. General Statements
We are grateful to the three Reviewers for their detailed assessment of our manuscript and are delighted about their very constructive and positive evaluations, highlighting the study’s novelty and rigor.
Briefly, the main points raised by Reviewers 1 and 3 do not involve additional experiments and are mostly about rethinking manuscript structure (e.g. moving data/analyses to the supplement or removing them altogether, as they distract from the main thrust of the story) and making the text overall less dense and more readable.
Reviewer 3 also raises a number of additional interesting points that we should discuss in our manuscript, which would allow us placing our findings more effectively into the context of the existing literature.
All these points are very well taken and will be implemented (see below, under 2).
Reviewer 2 is overall also rather positive – speaking of “a very careful and detailed study that addresses an important issue” and the study being “really rigorous and the logic […] very well explained”; moreover, this Reviewer also shares the view of both other Reviewers that parts of the manuscript (i.e., in particular its beginning) should be shortened.
Importantly, this Reviewer remarks in addition under “Significance”: “Without additional mechanistic insights suggesting that there is something particular different about the regulation of these mRNAs the manuscript is not of extremely high significance.” – an important point of criticism that we wish to address in our revision, as detailed below.
2. Description of the planned revisions
In the following, we detail how we plan to address the points raised by the Reviewers. The order in which we treat the points follows their – in our view – relative importance according to the Reviewers’ feedback. In particular the first item below, under (A), is the main point of criticism that we feel we should address carefully for the future revised version.
(A) Major point raised by Reviewer 2: “However, the study falls short on addressing the mechanism of this regulation and if it is different of other feeding regulated mRNA oscillations. This diminishes the significance of the study unless additional mechanistic details are provided.” , which is cross-commented both by Reviewer 1: *“More importantly, clues to the mechanism (e.g. iron, heme) regulating the rhythmic translation of IRP1 and IRP2 IRE-mRNAs in liver would increase the significance of the work.” *as well as by Reviewer 3: “Reading the comment from Reviewer #2 over the lack of a mechanism to explain why only four transcripts with IREs amongst a larger pool are subject to circadian regulation by IRPs somehow reduces the significance of the study, one has to agree that a discovery - likely another component in the system - is wanting. I remain of the view that the present work exposes this "weakness" of the entire field in a global as opposed to a partial manner and in doing so, makes a significant contribution, especially by further sub-classifying the IRE-containing transcripts according to their responsiveness in the diurnal occupancy of their IREs.”
Our response and revision plan: Indeed, in the original version of our manuscript we established the link to feeding, yet we did not pinpoint the precise molecular cue that could underlie the rhythmic regulation observed on certain IRE-containing mRNAs. We did discuss the molecular candidates quite extensively in the Discussion section of the manuscript (Fe2+; oxygen; reactive oxygen species), and it remains quite obviously the main question whether the observed diurnal control could be mediated directly by changes in intracellular iron availability.
Of note, the preprint by Bennett et al., for which we cite the initial biorXiv version in our manuscript, was updated very recently (https://doi.org/10.1101/2023.05.07.539729 – see version submitted December 18, 2023). It now includes new data that analyses around-the-clock iron levels also in liver. Briefly, the preprint shows, first, that serum iron is rhythmic with a peak during the dark phase at ZT16 (Figure 1D in Bennett et al.) yet loses rhythmicity when feeding is restricted to the light phase (Bennett et al., Figure 2E), indicating both feeding-dependence and circadian gating. Moreover, liver total non-heme iron – quantified using a method that measures both ferrous Fe(II) and ferric Fe(III) – shows low-amplitude diurnal variations which, however, do not meet the threshold for rhythmicity significance (Bennett et al., Figure 3G). Still, the difference between timepoints ZT4 (lower iron; light phase) and ZT16 (higher iron; dark phase) is reported as significant, with a fold-change that is not very pronounced (not* compatible with the observed direction of regulation of* Tfrc* mRNA, whose higher abundance in the dark phase would rather be in line with *lower *cytoplasmic iron levels, as pointed out by the authors.
Thus, at first sight the analyses by Bennett et al. would appear to answer part of the Reviewer’s question and point towards other mechanisms of regulation than iron levels themselves. However, it should be pointed out that the particular methodology for iron measurements used by the authors includes the use of reducing reagents and hence quantifies the sum of Fe2+ and Fe3+ iron. Large amounts of iron are stored in the liver in the form of ferritin-bound Fe3+, yet the bioactive, low-complexity iron that is considered relevant for IRP regulation is in the Fe2+ form. Therefore, the question whether bioactive ferrous iron levels follow a daily rhythm, compatible with the observed IRP/IRE rhythms described in our manuscript, still remains an open question and warrants a dedicated set of experiments that we are proposing to conduct in response to the Reviewers’ comments.
Briefly, for the revision we propose to use liver pieces from the two relevant timepoints of our study (i.e., ZT5 and ZT12) and apply a method that allows the separate quantification of Fe2+ and Fe3+ (Abcam iron assay ab83366; this assay can be adapted to liver iron measurements, see e.g. PMID31610175, Fig. 4A). This experiment will provide novel and decisive data on the molecular mechanism that may regulate the IRP/IRE system in a rhythmic fashion and therefore add to the significance of our findings, as requested by the reviewers.
Moreover, we believe that the outcome of the experiment would be very interesting either way, i.e. if we find rhythms in Fe2+ that are compatible with rhythmic IRP/IRE regulation, we would be able to provide excellent evidence in term of likely molecular mechanism and rhythmicity cue. If, by contrast, we find that Fe2+ is *not *rhythmic, it will point towards a mechanism that is distinct from simple Fe2+ concentrations.
In the latter case, collecting additional evidence on relevant alternative molecular cues would be beyond our capabilities for this particular manuscript, as it would require quite sophisticated methodological setup and preparation. For example, one could imagine that measuring around-the-clock liver oxygen levels* in vivo* – another candidate cue – would be highly interesting, yet we would not be able to conduct these experiments in a reasonable time frame (to start with, we would first need to request ethics authorisation from the Swiss veterinary authorities, which would in itself take ca. 4-6 months before we could even start an experiment). Thus, in the case of non-rhythmic iron levels, we would leave the question of other responsible cues open, but still think that with a balanced discussion of the resulting hypotheses we could provide significant added value to our work.
(B) Major comment raised by Reviewer 1: “Alas2 is expressed mainly in erythroid cells and not liver, whereas Alas1 is ubiquitously expressed. Therefore, it is possible that Alas2 in this study may originate from red cells/reticulocytes in the liver, and not from hepatocytes.”
Our response and revision plan: We would like to thank the Reviewer for the comment that is indeed pertinent. It is well established that Alas1 is the main transcript encoding delta-aminolevulinate synthase activity in hepatocytes, and Alas2 is about 10-fold less abundant in total liver RNA-seq data (quantified form own RNA-seq data, not shown).
We are nevertheless relatively sure that the Alas2 signal comes from low expression in hepatocytes; the best argument in support of this hypothesis is the analysis of single-cell RNA-seq data, as shown in the following Revision Plan Figure 1, which we would be happy to include in a revised version of the manuscript if the reviewers wish:
(C) Minor comment raised by Reviewer 1*: “The paper is dense and not easy to read. For example, the section on Tfrc regulation and NMD regulation is lengthy and perhaps not necessary for the paper and the section on "Previous observations in IRE-IRP regulation...." could be included in the discussion rather in than in the Results section. Some figures could be included in a supplement.” *continued in Referee cross-commenting “I agree with Reviewer 2 that the first sections in the manuscript are lengthy and not needed.”; moreover, Reviewer 2: “Also, the manuscript first sections (which mainly describe negative results) seem too long and descriptive.”
Our response and revision plan: We shall reorganize the paper accordingly, with the aim of making it an easier, shorter, clearer read. Many thanks for the input.
(D) Minor comment raised by Reviewer 1: “A description of the new anti-IREB2 antibody is needed. What IRP2 sequence was used to generate antibodies?”
Our response and revision plan: The following information will be included in the manuscript: “Rat monoclonal antibodies against ACO1/IRP1 and IREB2/IRP2 were generated at the Antibodies Core Facility of the DKFZ. Briefly, full-length murine ACO1/IRP1 and IREB2/IRP2 proteins, fused to a poly-histidine tag, were expressed in E. coli and purified on Ni-NTA columns using standard protocols. Purified His-tagged proteins were used to immunize rats and generate hybridomas. Hybridoma supernatants were first screened by ELISA against His-tagged ACO1/IRP1 and His-tagged IREB2/IRP2. As an additional control, supernatants were tested against full-length His-tagged murine ACO2 (mitochondrial aconitase), which shares 27 and 26% identity with ACO1/IRP1 and IREB2/IRP2, respectively. Supernatants reacting specifically with ACO1 or IREB2 were validated by western blotting using extracts from wild-type versus ACO1- or IREB2-null mice.”
(E) Minor comment raised by Reviewer 1: “A model summarizing the data would be useful.”
*__Our response and revision plan:__ Thank you for the suggestion – this will be done.
(F) “Optional” idea raised by Reviewer 3: “One nuance in the field of circadian biology is that a rhythm is deemed to be genuinely "circadian" when it continues in the absence of zeitgebers. In this sense, although all experiments are valuable, the "collapse" of the rhythm in the paradigms where dietary rhythms have been disrupted makes the phenomenology a candidate "epiphenomenon" rather than being closer related to the biological clock(s). Likewise, in the manuscript we never learn how the liver IRE-binding activity behaves in constant darkness.”
Our response and revision plan: This is an important aspect that we can clarify more specifically in our manuscript. It is true that constant (darkness) conditions are used to call a phenomenon circadian. We would nevertheless argue that for a rhythmic feature that is specifically found in liver, the constant darkness definition to distinguish circadian from non-circadian is not fully valid because even in constant darkness, the liver clocks are *not *in a free-running state but continue to be entrained by the SCN clock (it is only the latter that is free-running under these conditions).
In our manuscript, we actually suggest that the observed rhythms are not a core output of the circadian machinery (Fig. 6 of our manuscript), but indirectly engendered through feeding rhythms, which are coupled to sleep-wake cycles and thus connect in an indirect way to the central circadian clock activity in the SCN.
In wild-type mice we would therefore expect that irrespective of constant darkness or light-dark entrainment (and assuming *ad libitum *feeding), the hepatic rhythms of the relevant IRE-containing transcripts would persist in a similar fashion.
(G) “Optional” idea raised by Reviewer 3: “Where the authors mention in a parenthesis "moreover, there are documented links between iron and the circadian timekeeping mechanism itself", I invite them to take a closer look to the paper Konstantinos Mandilaras and I coauthored in 2012 "Genes for iron metabolism influence circadian rhythms in Drosophila melanogaster". In that work, we showed that RNA interference of genes that are required for iron sulfur cluster formation (including on IRP1) in the central clock neurons of the fly result in loss of the circadian rhythm when flies were kept at constant darkness (not so when they were kept under light:dark oscillation). So this point should probably remain open..”
Our response and revision plan: We would like to thank the Reviewer for pointing out this interesting connection that would fit well into the context of our manuscript. It should be cited in the context of our current Figure 3, where we measure in vivo and in tissue explants whether IRP-deficiency affects the clock itself.
To follow Reviewer 3’s idea, we have gone a little further in our analyses of around-the-clock expression data to see if any of the components of the Fe-S assembly machinery is rhythmic itself, which could have the potential to add novel information.
Briefly, we have used for this purpose our around-the-clock RNA-seq and ribo-seq data from PMID 26486724. In summary, we find that the expression at RNA and/or footprint level is non-rhythmic for the vast majority of genes involved in FeS biogenesis, assembly or transport, with the exception of low-amplitude rhythms for Glrx5 and Iba57 (Revision Plan Figure 2).
By contrast, all of the following other genes are non-rhythmic throughout (list of Fe-S-relevant genes from PMID34660592): Cytoplasmic/nuclear, all non-rhythmic:* Cfd1=Nubp2, Nbp35=Nubp1 , Ciapin1, Ndor1, Iop1=Ciao3=Narfl, Ciao1, Ciao2b=Fam96b, Mms19, Ciao2a=Fam96a; mitochondrial, all non-rhythmic: Iscu, Nfs1, Isd11=Lyrm4, Acpm=Ndufab1, Fdx1, Fdx2=Fdx1l, Fxn, Hspa9 Hsc20=Hscb, Abcb7, Alr=Gfer, Isca1, Isca2, Nfu1*
As these are mainly “negative results”, and as we are also unable to propose a solid possible mechanistic connection between the Glrx5 and/or Iba57 rhythms and the rest of the story of our manuscript, we do not intend to include such data in our manuscript, but are only putting it for the record into this rebuttal.
3. Description of the revisions that have already been incorporated in the transferred manuscript
NONE
4. Description of analyses that authors prefer not to carry out
NONE – we think we can address all points as described above.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The present manuscript highlights the previously neglected component of diurnal rhythms into the study of iron regulation in the liver, a key organ in the systemic regulation of the metal. A major, well substantiated finding is that IRP1 takes over from IRP2 as a highly relevant regulator during a time of maximal use of the combined system. The presence of a "dietary signal" that sustains the cycling of cellular IRE binding activity in the liver, although undisputable, is perhaps a lesser claim until these or other investigators can confirm or refute the possibility that iron itself (i.e., the best-established factor affecting …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The present manuscript highlights the previously neglected component of diurnal rhythms into the study of iron regulation in the liver, a key organ in the systemic regulation of the metal. A major, well substantiated finding is that IRP1 takes over from IRP2 as a highly relevant regulator during a time of maximal use of the combined system. The presence of a "dietary signal" that sustains the cycling of cellular IRE binding activity in the liver, although undisputable, is perhaps a lesser claim until these or other investigators can confirm or refute the possibility that iron itself (i.e., the best-established factor affecting cellular IRE-binding) is such a signal. If iron does the job, then the interest lies in showing diurnal rhythmicity of its availability in the circulatory system, presumably linked to dietary iron absorption. There is plenty of clinical evidence of this in humans, but the present study along with the cited preprint by Bennett et al. appear to be the first demonstrations of this diurnal variation in mice.
The manuscript has other strengths not immediately evident from the above claims made in the abstract. It contains an elegant balance of reanalyzing and reassessing "big data" produced by the same laboratory in the past in light of new experimental findings that have appeared in the meantime in the literature together with important new additions of such data from combined RNAseq and ribo-seq collections using IRP1 and IRP2 knockout animals and, importantly, by making use of published material from other studies that have provided relevant comparators from other knockouts that studied aspects of liver circadian biology. The approach, besides providing robust testing of the ideas presented, opens the field to questions that remain unanswered but seem highly relevant (I come to these below). The authors write in a very open manner not only about the new findings but also about aspects we do not understand, and their systems biology approach is likely to generate new hypotheses to address incognita.
Let me therefore be clear upfront that the response that follows is written on the premise that I evaluate the work presented as ready for publication: The figures have been constructed with care summarizing a lot of careful investigations and the main conclusions derive seamlessly from the experimental data. Rather than taken as potential criticism to the authors, I would ask that the counterviews or limitations that may arise from my response to the paper are better taken as a celebration of the work - and views - presented. Such a discussion is only possible due to the open style of this communication mentioned above and is meant to provoke a dialogue or even drive further questioning of the datasets and the design of future experimental approaches. If the authors find any of these comments useful for their revision, they are welcome to take them onboard, but everything that follows should be read under the term "optional".
One nuance in the field of circadian biology is that a rhythm is deemed to be genuinely "circadian" when it continues in the absence of zeitgebers. In this sense, although all experiments are valuable, the "collapse" of the rhythm in the paradigms where dietary rhythms have been disrupted makes the phenomenology a candidate "epiphenomenon" rather than being closer related to the biological clock(s). Likewise, in the manuscript we never learn how the liver IRE-binding activity behaves in constant darkness. Where the authors mention in a parenthesis "moreover, there are documented links between iron and the circadian timekeeping mechanism itself", I invite them to take a closer look to the paper Konstantinos Mandilaras and I coauthored in 2012 "Genes for iron metabolism influence circadian rhythms in Drosophila melanogaster". In that work, we showed that RNA interference of genes that are required for iron sulfur cluster formation (including on IRP1) in the central clock neurons of the fly result in loss of the circadian rhythm when flies were kept at constant darkness (not so when they were kept under light:dark oscillation). So this point should probably remain open.
Given the phenotypes collected from RNAi in different cell types, we became sensitive to the notion that different cell types may work with different sets of what we might call collectively "iron metabolism genes". Thus, while reading the present differences between the relative contributions of IRP1 and IRP2 in mouse liver at different times during the day (and night), I kept wondering if both function in the hepatocytes or whether the macrophages or other cell types in the liver may have their own particular contributions.
Another issue raised by the authors early on relates to the differential effects of IRP1/2 "activation" on different IRE-containing transcripts. This is a fascinating problem, not answered by the six transcripts shown in figure 1G, but I consider that the present paper offers a great service to the field in figure 5I, where an even more comprehensive grouping of IRE-containing transcripts is provided in terms of their "regulation" by IRPs. Future research should attempt to discover features that correlate within the sets of transcripts, as grouped in here.
Thus, another point made repeatedly by the authors that despite four decades of work on the IRPs we still have open questions about how they "regulate" is well taken. In the same tone, their results in relation to IRP2 degradation show that the story of how the presence of iron leads to the degradation of IRP2 has not been fully elucidated, either.
Referees cross-commenting
Reading the comment from Reviewer #2 over the lack of a mechanism to explain why only four transcripts with IREs amongst a larger pool are subject to circadian regulation by IRPs somehow reduces the significance of the study, one has to agree that a discovery - likely another component in the system - is wanting. I remain of the view that the present work exposes this "weakness" of the entire field in a global as opposed to a partial manner and in doing so, makes a significant contribution, especially by further sub-classifying the IRE-containing transcripts according to their responsiveness in the diurnal occupancy of their IREs.
I would like to reinforce the comment of reviewer 1 with respect to the antibodies used in this study that should be made available to the community, given the specificity described. My congratulations to the authors.
Significance
A lesson, perhaps, for the field is that sometimes more than one mechanism may be at play in different cellular or physiological contexts, while vigorous testing requires time and resources and we should value examples of such care and openness, an example of which is offered, in my view, by the present study.
Fanis Missirlis
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In the manuscript entitled "Diurnal control of iron responsive element (IRE)-containing mRNAS through iron regular proteins IRP1 and IRP2 is mediated by feeding rhythms", Nadimpalli et al. uncover and mechanistically dissect how the circadian clock and feeding regulates the expression of proteins involved in iron homeostasis in mice. The authors first utilized RNAseq and ribose data and found that a subset of mRNAs containing IREs display rhythmic translation in the liver and/or kidney. The authors then utilized previously published or newly generated datasets to study the origin of these oscillations. After a careful and thoughtful …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In the manuscript entitled "Diurnal control of iron responsive element (IRE)-containing mRNAS through iron regular proteins IRP1 and IRP2 is mediated by feeding rhythms", Nadimpalli et al. uncover and mechanistically dissect how the circadian clock and feeding regulates the expression of proteins involved in iron homeostasis in mice. The authors first utilized RNAseq and ribose data and found that a subset of mRNAs containing IREs display rhythmic translation in the liver and/or kidney. The authors then utilized previously published or newly generated datasets to study the origin of these oscillations. After a careful and thoughtful examination, they determine that the oscillations of those mRNAs in the liver are mainly driven by feeding-associated signals, although they are influenced by other factors. This is a very careful and detailed study that addresses an important issue. The study is really rigorous and the logic is very well explained. So overall this study is very solid and the main conclusion of the study (that the oscillations of those mRNAs are driven by feeding) is solidly established. However, the study falls short on addressing the mechanism of this regulation and if it is different of other feeding regulated mRNA oscillations. This diminishes the significance of the study unless additional mechanistic details are provided. Also, the manuscript first sections (which mainly describe negative results) seem too long and descriptive. Still this is an important and solid study.
Significance
The main issue this reviewer has with the manuscript is the significance. Without additional mechanistic insights suggesting that there is something particular different about the regulation of these mRNAs the manuscript is not of extremely high significance.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
This paper provides evidence for the diurnal regulation of specific subset of iron regulatory elements (IREs)-containing mRNAs in liver by iron regulatory proteins 1 and 2 (IRP1 and IRP2) in mice. The authors show that IRP2 oscillates over 24 h period to regulate IRE-containing mRNAs in the light phase, and collaborates with IRP1 to regulate IRE-mRNAs in the dark phase.
Major Comments
The authors have carefully performed experiments, and convincingly show that 5'-IRE containing transcripts (Fth1, Ftl1, Fpn and Alas2) display significant amplitude rhythms in ribosome occupancy in liver. Tfrc mRNA, which harbors a 3' IRE, …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
This paper provides evidence for the diurnal regulation of specific subset of iron regulatory elements (IREs)-containing mRNAs in liver by iron regulatory proteins 1 and 2 (IRP1 and IRP2) in mice. The authors show that IRP2 oscillates over 24 h period to regulate IRE-containing mRNAs in the light phase, and collaborates with IRP1 to regulate IRE-mRNAs in the dark phase.
Major Comments
The authors have carefully performed experiments, and convincingly show that 5'-IRE containing transcripts (Fth1, Ftl1, Fpn and Alas2) display significant amplitude rhythms in ribosome occupancy in liver. Tfrc mRNA, which harbors a 3' IRE, also showed a rhythmic pattern in both liver and kidney. The changes in IRE-containing mRNAs correlated with IRP2 protein abundance. Further studies performed using Aco1 and Ireb2 knockout mice showed that both IRP1 and IRP2 are required for rhythmic regulation of IRE-containing mRNAs. Overall, the findings in this paper are interesting and novel, and show for the first time that IRE-containing mRNAs required for maintenance of cellular iron metabolism and IRP2 are subjected to rhythmic regulation. Alas2 is expressed mainly in erythroid cells and not liver, whereas Alas1 is ubiquitously expressed. Therefore, it is possible that Alas2 in this study may originate from red cells/reticulocytes in the liver, and not from hepatocytes.
Minor Comments
The paper is dense and not easy to read. For example, the section on Tfrc regulation and NMD regulation is lengthy and perhaps not necessary for the paper and the section on "Previous observations in IRE-IRP regulation...." could be included in the discussion rather in than in the Results section. Some figures could be included in a supplement. A description of the new anti-IREB2 antibody is needed. What IRP2 sequence was used to generate antibodies? A model summarizing the data would be useful.
Referees cross-commenting
I agree with Reviewer 2 that the first sections in the manuscript are lengthy and not needed. More importantly, clues to the mechanism (e.g. iron, heme) regulating the rhythmic translation of IRP1 and IRP2 IRE-mRNAs in liver would increase the significance of the work. Overall, the findings are novel, and would be of interest to the iron metabolism and circadian rhythm fields.
Significance
Previous studies have reported a role for iron in altering gene expression and circadian rhythms in mice. The current manuscript extends these studies to show that several IRE-containing mRNAs in liver and IRP2 are subjected to rhythmic regulation. These findings will be interest to researchers in circadian rhythm and iron metabolism fields.
-