Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during the mitosis-to-G1 phase transition
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Abstract
Accurate control of the cell cycle is critical for development and tissue homeostasis, and requires precisely timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle in human cells. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and we identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.
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Referee #3
Evidence, reproducibility and clarity
In this paper, Dr. Tanenbaum and colleagues present a new method to determine the exact point of the cell cycle in which a cell is. This new method has the potential to be applied to any cell-cycle phase and even to other processes. Focusing on M-G1 transition, they do sequencing of single human cells. They identify two groups of cell cycle-related genes, expressing mRNAs which are degraded either immediately after exit from mitosis or later on during G1. One of the factors involved in this degradation is identified as CNOT1.
In my opinion, the new method is well stablished and has the potential to be very useful to future work. In …
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Referee #3
Evidence, reproducibility and clarity
In this paper, Dr. Tanenbaum and colleagues present a new method to determine the exact point of the cell cycle in which a cell is. This new method has the potential to be applied to any cell-cycle phase and even to other processes. Focusing on M-G1 transition, they do sequencing of single human cells. They identify two groups of cell cycle-related genes, expressing mRNAs which are degraded either immediately after exit from mitosis or later on during G1. One of the factors involved in this degradation is identified as CNOT1.
In my opinion, the new method is well stablished and has the potential to be very useful to future work. In general, I think that the main conclusions are based on more than one approaches and are convincing. In relation to these, I have two major concerns:
- Although it is clear that a scheduled mRNA decay exits, this does not exclude the possibility of a concomitant effect on mRNA synthesis. A measurement of nascent transcription is needed.
- As mentioned in the discussion, it is possible that the limited effect of depleting CNOT1 is due to the partial knockdown. However, it is also possible that a different pathway of mRNA degradation is involved. This should be addressed by targeting other decay factors (for example Xrn1 and/or an exosome component).
Minor comments:
- Cell cycle control is not absolutely universal. The authors should mention that their results and conclusions correspond to human cells; if not in the title, at least in the abstract.
- Is SORT-seq (mentioned exclusively in the methods section) the same as scRNA-seq?
- Figure 2: there is no correspondence between the TOP2A images and their quantification. This experiment also needs an unrelated mRNA FISH as a negative control.
- Figure 3D: if I correctly understood this experiment, "Time in Actinomycin D" is a better title for the X axis. "Time after mitotic shake-off" is misleading because it suggests that the cells were released from the mitotic blockage.
- Figure S3L: the blockage of transcription by Actinomicin D should be demonstrated by a more general method, such as EU (5-ethynyl uridine) incorporation.
- Figure 4A: what is the cell cycle phenotype of CNOT1 siRNA? If a reduction with CRISPRi of 50 % causes a phenotype (Figure S4C), then a reduction of 90 % by the siRNA (Figure S4A) should cause a stronger phenotype.
- Do the two waves of protein degradation (mentioned in the second paragraph of page 12) affect the same genes than the two waves of mRNA degradation?
Significance
The new method described in this paper could be useful to address many questions in the field of cell-cycle control. Moreover, with modifications, it could be applied to any time-dependent biological problem. In addition, the scheduled degradation of mRNAs in the M-G1 transition is a discovery of biological significance.
In my opinion, the paper is technically interesting to a broad range of investigators, and biologically relevant for those studying the cell cycle.
Keywords of my field of expertise: transcription, chromatin, gene expression.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study Krenning, Sonneveld, and Tanenbaum have investigated the temporal control of mRNA decay after mitosis. Previous work has demonstrated that following mitosis, regulated protein degradation serves an important role in erasing any lingering memory of the previous cell cycle. Left unanswered however, is whether mRNA decay is similarly regulated after mitosis, and if so, what role does it play in the cell cycle. The authors use time-lapse imaging and single-cell RNAseq to measure decay rates of mRNAs after mitosis. They use the FUCCI sensors to identify the precise age of each individual cell prior to performing …
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study Krenning, Sonneveld, and Tanenbaum have investigated the temporal control of mRNA decay after mitosis. Previous work has demonstrated that following mitosis, regulated protein degradation serves an important role in erasing any lingering memory of the previous cell cycle. Left unanswered however, is whether mRNA decay is similarly regulated after mitosis, and if so, what role does it play in the cell cycle. The authors use time-lapse imaging and single-cell RNAseq to measure decay rates of mRNAs after mitosis. They use the FUCCI sensors to identify the precise age of each individual cell prior to performing single-cell RNAseq. Using this pseudo-timelapse approach, they identify several genes that are actively degraded after mitosis. Interestingly, they find that some mRNAs are decayed rapidly and immediately after mitosis while other mRNAs are decayed only after a delay of about 1.5 hours after mitosis. The authors find that, at least for several of the genes, the protein CNOT1 mediates the decay. The authors conclude that regulated mRNA decay occurs after mitosis and helps reset the transcriptome at the onset of a new cell cycle.
The question being asked in this manuscript is interesting and potentially very important to the cell cycle field. However, I believe this study suffers from several technical issues highlighted below as well as relatively modest effect sizes, particularly when it comes to ascribing CNOT1 as the key regulator mediating mRNA decay upon mitotic exit.
Major Comments:
The authors repeatedly claim that they can accurately measure the time a cell has spent in G1 phase, due to using live-cell imaging to calibrate their FACS data. However, following mitosis, cells bifurcate into either G0/Quiescence or into G1 phase. Importantly, the FUCCI-G1 sensor is not capable of distinguishing between these two cell cycle phases. Thus, while two cells may have spent the same amount of time since mitosis, one cell may be in Quiescence while the other cell is in G1 phase and they would both have the same level of FUCCI-G1. Even by live-cell imaging its not possible to distinguish between these two cell cycle phases with the markers used in this study. I believe you can see evidence for this in the Flow cytometry data in Figure 1C where there is a large population of cells with very high FUCCI-G1 levels (even higher than most of the S phase cells) but very low FUCCI-G2 levels. These cells likely represent cells that entered G0 after mitosis, remained there for several hours, and thus accumulated very high levels of the FUCCI-G1 sensor. This technical limitation has several implications. First, it means what is likely going into the single-cell mRNA seq workflow is a mixture of G0 and G1 cells. Since the authors observe two distinct "waves" of mRNA decay, it makes me wonder if one wave might be occurring in G1 cells while the other wave is occurring in G0 cells? Second, the authors state several times they can accurately determine the time a cell has spent in G1 phase. The more accurate statement however is that the authors can accurately determine the time that has elapsed since mitosis, since they cannot distinguish between G0 and G1 phase. This is admittedly a nuanced distinction but an important one given that quiescence cells are in a very different cellular state than cells in G1 phase. The authors actually use the correct x-axis label of "Time after metaphase (min)" in several of their figures, but they do not use the same language in the main text or to describe their conclusions. To overcome the technical challenge of distinguishing between G0/G1 cells and to address these two points, the authors could use an additional FUCCI sensor, mVenus-p27K(-), which accumulates in G0 cells but not in G1 cells (PMID: 24500246).
One of the main novelty claims of the paper is that mRNA decays following mitotic exit in two waves. In order to support this claim, the authors plot the time since metaphase vs normalized transcripts for many single cells. For several genes including CDK1, TOP2A, and UBE2C, there is an immediate drop from time 0 to the next most densely populated timepoint of about 20 minutes. This results in a bimodal histogram as seen in Figure 2A, where there is one single bin representing genes that are maximally decayed at time 0, while another population is more normally distributed with a median decay time of ~80minutes. I have two concerns about this data. First, there are very few if not zero cells analyzed that were between 0 and 20 minutes old at the time of collection. Therefore the authors do not know if the mRNA decayed immediately after mitosis or with a 20minute delay after mitosis. Thus, rather than some genes decaying immediately after mitotic exit resulting in that one bin at time=0 and some genes decaying with a delay, there could actually be just one wave of mRNA decay that is broadly and normally distributed from 20-80minutes after mitosis (see histogram Figure 2 and Figure S3G; if you ignore anything less than 20minutes due to lack of data during this time window, then there is just a single distribution). While likely technically challenging due to cytokinesis, sampling cells that are between 0-20minutes old may be important to accurately measuring the decay rates of mRNAs upon mitotic exit. Second, its not entirely clear from the methods section, but I am assuming that for time 0 of the plots in Figure S3A-F, the authors are using G2/M sorted cells as defined by FUCCI-G2 high/FUCCI-G1 low status. However, depending on what precise FACS gates were used to do this sort, these cells could be anywhere in early G2, late G2, prometaphase, metaphase, etc and they are all averaged together. Thus, when the authors claim that the mRNAs are decaying immediately after mitosis, that's based on comparing the mRNA levels in this mixed G2/M population with cells that are 20minutes or more after mitosis. For genes like CDK1 where the mRNA is almost completely gone at 20 minutes after mitosis when they first have any cells to measure, its entirely possible these mRNAs were already decayed either before or within mitosis, rather than upon mitotic exit. What we really need to know is what are the mRNA levels precisely at metaphase and plot that value as time=0 (for example in figure S3A-F). Perhaps the authors could use CDK1i plus Taxol treatment to accumulate cells in mitosis like they describe in the methods for the transcription inhibition experiments. (If this is what was done for the Sort-Seq experiments as well as the data in Fig S3A-F, my apologies for not completely understanding, but the methods section should be updated to make this more clear)
As the authors point out in the discussion, the effect size of CNOT1 depletion on mRNA decay are rather small. Notably the genes that appear to be statistically significant are all the genes that are hardly decayed after mitosis anyways (see Figure 4D, ARLGIP1, PSD3, etc). These genes appear to be only down about 2-fold (-1 on a log2 scale axis) in the control group, which would be expected during mitosis when the mother cell splits in half into two daughter cells. Depending on the method of normalization, this change in mRNA levels may simply be due to this. I understand the technical limitations in knocking down an essential gene, but given that one of the major novelty claims of this study is that CNOT1 mediates the mRNA decay upon mitotic exit, more robust proof is warranted. The authors could employ the inducible protein degradation system they referenced in the discussion. Additionally they could perhaps show data that CNOT1 is somehow differentially regulated during mitotic exit that would account for this sudden change in mRNA stability. If the authors were to include additional corroboratory data demonstrating CNOT1 is most active during mitotic exit for example, it would help to overcome that low effect size upon mild CNOT1 depletion.
Minor comment:
The authors state that they can "accurately determine the time a cell has spent in G1 phase based on its FUCCI-G1 fluorescence as measured by FACS" (Page 6, last line of the first paragraph). It would be nice to know how accurately? What is the level of uncertainty in your measurements? +/- how many minutes?
The authors only focused on those genes whose mRNA's were decayed upon mitotic exit, but of equal interest would be those cell cycle genes that were not observed to decay upon mitotic exit. The authors may want to highlight these genes, because it might shed light on what aspects of the cell cycle cells want to reset, and what aspects of the cell cycle the cells may wish to "remember" from the previous cell cycle. For example, what is the status of the cyclins? CIPs? Origin licensing genes? There is growing evidence that the status of the mother cell can influence the cell cycle of the daughter cell and mRNA levels of key cell cycle genes have been implicated in this (PMIDs: 28514656, 28317845, 28869970).
Significance
This paper offers a potential conceptual advance to our understanding of how cells reset after the cell cycle is completed. By demonstrating that cells regulate protein as well as mRNA degradation upon cell cycle exit, the authors show cells utilize multiple mechanisms to reset the biochemical state of the cell at the onset of a fresh cell cycle. This work would be of potential interest to the cell cycle field, particularly those interested in G1 regulation. It would also be of interest to people interested in mRNA regulation as it would demonstrate a clear window during which mRNA decay was specifically upregulated.
While my expertise lies in the cell cycle and the use of FUCCI sensors to identify cell cycle stages, I have less expertise in single-cell RNAseq and methods of mRNA quantification.
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Referee #1
Evidence, reproducibility and clarity
In the manuscript "Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during mitotic exit", Krenning et al. describe a method that connects live-cell microscopy with single-cell RNA sequencing in order to monitor global changes in mammalian mRNA gene expression in a cell-cycle-dependent manner. To this end they employ a fluorescent, ubiquitination-based cell cycle indicator (FUCCI system) in human untransformed RPE-1 cells coupled to SORT-Seq in order to generate a high-resolution, time-resolved transcriptome profile of cells spanning the transition form M phase into G1 phase. By comparing FACS-based …
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
In the manuscript "Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during mitotic exit", Krenning et al. describe a method that connects live-cell microscopy with single-cell RNA sequencing in order to monitor global changes in mammalian mRNA gene expression in a cell-cycle-dependent manner. To this end they employ a fluorescent, ubiquitination-based cell cycle indicator (FUCCI system) in human untransformed RPE-1 cells coupled to SORT-Seq in order to generate a high-resolution, time-resolved transcriptome profile of cells spanning the transition form M phase into G1 phase. By comparing FACS-based sampling with an in silico trajectory inference method the authors provide convincing evidence that this system allows for high-resolution, time-resolved transcriptome profiling of cells in the transition from M-phase into G1 phase. Subsequent analysis of changes in steady-state gene expression revealed a set of >200 transcripts that undergo rapid decay in two distinct waves, first around the time of mitotic exit and second upon G1 entry. Those results were independently validated by single molecule FISH of select transcripts, revealing that the first wave of RNA decay initiates at the start of anaphase and the second wave starts during early G1 phase. Using mathematical modeling followed by selective validation (using cell-cycle staging combined with global inhibition of RNA decay), the authors derive and confirm precise mRNA decay rates for cell cycle regulated mRNAs that were overall lower than what has been observed in previously described population-based half-life measurements. Finally, the authors establish a role of deadenylation by the CCR4-NOT complex in selective mRNA decay during M-phase/G1 transition by revealing a partial stabilization upon NOT1-depletion by RNAi.
I have only a minor comment to help improve the mechanistic insights: While the authors attribute the partial stabilization of cell-cycle regulated mRNAs to an incomplete depletion of NOT1 (which is possible), an alternative hypothesis would be that decapping and 5´-to-3´ decay further contribute to mRNA turnover. Notably, the authors describe the use of siRNAs targeting Dcp2 in the methods section (without referring to it in the results part), indicating that they may have data that could clarify this. They authors may consider adding this data to complete an otherwise nicely executed and well-written manuscript.
Significance
Overall, this is an impressive and well-controlled body of work providing novel insights into cell-cycle controlled mRNA decay. It reveals novel insights into the targets and molecular mechanisms of mRNA decay in the transition of mitosis to G1, thereby adding significantly to our understanding of the scope of tightly controlled gene expression for proper execution of the cell cycle.
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