Nuclear pore complex acetylation regulates mRNA export and cell cycle commitment in budding yeast

  1. Mercè Gomar‐Alba
  2. Vasilisa Pozharskaia
  3. Bogdan Cichocki
  4. Celia Schaal
  5. Arun Kumar
  6. Basile Jacquel
  7. Gilles Charvin
  8. J Carlos Igual
  9. Manuel Mendoza

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Nuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm, and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyltransferases (KATs) promote transcription through acetylation of chromatin‐associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX‐2) complex, to the nuclear basket. The Esa1‐mediated nuclear export of mRNAs in turn promotes entry into S phase, which is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism is not only limited to G1/S‐expressed genes but also inhibits the expression of the nutrient‐regulated GAL1 gene specifically in daughter cells. Overall, these results reveal how acetylation can contribute to the functional plasticity of NPCs in mother and daughter yeast cells. In addition, our work demonstrates dual gene expression regulation by the evolutionarily conserved NuA4 complex, at the level of transcription and at the stage of mRNA export by modifying the nucleoplasmic entrance to nuclear pores.

Article activity feed

  1. Version published to 10.15252/embj.2021110271
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    We thank all reviewers for their thorough assessment and constructive comments.

    For clarity, their comments have been numbered.

    __Reviewer #1 __

    Evidence, reproducibility and clarity:

    Summary:

    Acetylation/Deacetylation controls G1/s transition in budding yeast. The lysine acetyl transferase Esa1 is here shown to play a role, in part via acetylation of the nuclear pore complex basket component Nup60, which stimulates mRNA export.

    Major comments:

    1 • Figure 1C: The curve for esa1-ts in this figure and the curve in the supplementary figure S2B are not similar, while the first shows 10% cells budding after 60 minutes it is about 50% after 60 min in S2B. Another helpful way of presenting the data could be the length of the G1 phase (from cytokinesis to budding) in the WT, esa1-ts, gcn5delta cells over time.

    We thank the reviewer for pointing this out. Indeed, there is some day-to-day variability in the budding kinetics of the temperature-sensitive esa1 mutant, and the text referred to one individual experiment. Therefore, we have changed the text to better reflect the observed variability (p. 7) and added a graph (supplementary Figure S2C) including all individual replicates. This shows that in spite of small differences between experiments, esa1-ts cells always bud slower and less efficiently than wild-type cells. We note that the data cannot be shown in the way suggested (time from cytokinesis to budding, presumably from individual cells) because cells in these experiments were released from a G1 block (after cytokinesis), and samples from cell cultures were imaged at time intervals (and not single cells over time). Time-lapse data of single cells is shown in figure 2E.

    2 • What is the rational of creating the Nup60-KN mutation. Does it prevent acetylation of Nup60, at least by GCN5 and/or esa1?

    The biophysical properties of asparagine resemble those of acetylated lysine. Therefore, the Nup60-KN mutant (lysine 467 to asparagine) is expected to mimic acetylation of Nup60 K467, which was found to be acetylated in earlier studies. Supporting the conclusion that Nup60-KN is indeed an acetyl-mimic, the nup60-KN mutation partially rescues the Start and mRNA export defects on Esa1-deficient cells. We make the rationale of the Nup60-KN mutation clearer in the current version (p. 8).

    3 • Given the much stronger phenotype of the esa1-ts+GCN5 delta condition for G1/S transition as compared to esa1-ts and that GCN5 seems to strongly acetylate Nup60 I do not understand the sole focus on esa1 in the study. The fact that the Nup60-KN cells do not show G1/S transition under esa1-ts+GCN5 delta conditions in experiments presented in Fig. S3 argues that esa1 meaidted acetylation of Nup60 is only one, probably minor aspect of G1/S transition. This should be much balanced discussed.

    We focus on Esa1 because this allows us to dissect the specific role of Nup60 acetylation and mRNA export during the G1/S transition. Of course, Esa1-dependent acetylation of Nup60 is not the only process controlling the G1/S transition, which is regulated at several levels. For example, the concentration of multiple Start activators and inhibitors scales differentially with cell size (PMID: 26390151, 32246903). In addition, daughter-specific factors inhibit Start through a pathway parallel to Nup60 deacetylation (Ace2/Ash1-dependent repression of Cln3 transcription; PMID: 19841732, 19841732). We discuss these studies in the current version (p. 17).

    As for the relative contribution of Esa1 and Gcn5 to the G1/S transition and mRNA export: both of these KATs have overlapping roles in promoting transcription, probably through distinct substrates (such as histone H2 for Gcn5, H4 for Esa1) and this may contribute to their role in Start. Consistent with this, deletion of GCN5 causes a minor delay in transcription of G1/S genes (Kishkevich, Sci. Rep 2019). On the other hand, gnc5 mutants have no detectable mRNA export defects, unlike esa1-ts (our Figure 3E). This suggests that whereas Gcn5 and Esa1 may have overlapping roles in transcription of G1/S genes, Esa1 is more specifically involved in mRNA export. The ability of Nup60-KN to rescue the single mutant esa1 but not the double* gcn5 esa1* is consistent with this view: the transcription defects in the double mutant may be so severe as to prevent Start even in the presence of Nup60-KN. We have modified the discussion to mention these points. In addition, we will investigate the transcription defects of esa1 and gcn5 single and double mutants to test this possibility and include the results in a revised version.

    4 • Suppl: Fig 2: I miss the hat1delta+gcn5delta condition.

    We will include the budding index of the *hat1 gcn5 *double mutant in a revised version.

    Minor comments:

    5 • Figure legend 2C "at least 200 cells were scored": please state number of replicates

    Figure 2C shows RT-qPCR data. The reviewer probably means figure 1C, which shows the budding index of one experiment comparing wild type, esa1, gcn5 and esa1 gcn5 strains. This experiment was repeated 3 times, as is now mentioned in the figure 1 legend.

    6 • Figure 2E: X axis "impor" should be corrected to "import"

    We have corrected this.

    7 • Would Mex67 and/or Mrt2 overexpression recue the esa1-ts and esa1-ts+GCN5 delta phenotype?

    We will include this experiment in a revised version.

    8 • Figure 4 A: The size of the daughter cells in the hos3delta condition seems smaller as compared to esa1-ts. Is this true and can you comment this? Is a premature onset of S phase observed here?

    Since Fig 4A features only wild type and hos3∆ cells, the reviewer is probably referring to esa1-ts cells shown in figure 4B. These two figure panels are not directly comparable: cells in 4A are freely cycling, whereas those in 4B were released from a mitotic arrest using nocodazole. The mitotic arrest was done in order to avoid potentially confounding effects due to inactivation of Esa1 during S phase. However, the arrest also causes daughter cells to grow larger, explaining the size differences pointed out by the reviewer. That being said, it is true that cell size and G1 duration are intimately linked and thus the reviewer question raises a relevant point. We previously showed that although hos3 daughter cells enter S phase prematurely, their size is not significantly different from wild type (Kumar et al., Figure 1d-g). Premature onset of S phase can lead to smaller cell size but this is not the case for hos3 cells, probably due to the slightly faster growth rate of the hos3∆ mutant relative to wild type specifically during S/G2/M phases (Kumar et al., Supplementary Fig. 1b).

    9 • Figure 4D: The still images in figure 2E and 4D do not correspond with the quantitation. E.g. in Fig 2E the esa1ts cells shows Whi5 export at t=81 min, which is according to the shown quantitation unusual late.

    We will modify Figures 2E-4D in a revised version to include cells that export Whi5 at times closer to the median.

    10 • Figure 4B: it is not clear why for the quantitation a different representation is chosen as compared to 4A. It would be better to show the nuclear intensities of mother/daughter as in Figure 4A.

    The reason for the different representation between figures 4A and 4B is that 4A depicts freely cycling cells and in 4B, cells were released from a nocodazole-induced mitotic arrest (as mentioned in our response to point 8). A mitotic arrest perturbs M/D size asymmetries, as daughter cells (but not mothers) continue growing during the arrest, leading to larger nuclear size. In addition, esa1-ts daughters are smaller than wt daughters in this condition, further complicating M/D asymmetries. We thought that in this case, a better metric for protein association with the NPC is the fluorescence intensity relative to a nuclear pore component. We agree that using different types of graphs is confusing, and therefore we have removed M/D comparisons from figure 4A and now represent these data as in figure 4B: the intensity of Sac3 relative to Nup49. Finally, a good control for these experiments is the quantification of total protein levels, which we have added for Sac3. We have also removed Mtr2-GFP data until our analysis of Mtr2 total levels is complete. We hope this simplifies this figure.

    11 • Figure 4D: To strengthen these results, it would be good to perform this assay with esa1-ts Nup60-KN cells as in figure 2a. The release of Whi5-GFP is expected to behave in a similar way to the WT. This would ensure that Nup60 acetylation is a pre-requisite for Whi5 release

    I’m afraid we don't understand this suggestion. Figure 4D shows time-lapse fluorescence microscopy of Whi5 nuclear export when Sac3 is recruited to the nuclear basket. Figure 2a shows western blots of Nup60 acetylation status. Therefore it is not clear how these two assays could be done in similar ways. Perhaps the reviewer refers to a different figure panel. The purpose of the suggested experiment, if we understand properly, is to test whether Nup60 acetylation is required for Whi5 export. This is the hypothesis tested in figure 2D: Whi5-GFP export is delayed in esa1-ts, and this delay is partially rescued in esa1-ts nup60-KN, which mimics acetylation. In fact, the advance in Whi5 export observed in Figure 4D upon Sac3 anchoring to NPC is similar to that observed in a nup60-KN (Figure 2E).

    12 • Page 13 "Finally, we tested whether Esa1 targets Sac3 to G1 nuclei": The effect of esa1 knockdown on Sac3 fit with the story line and the effect esa1 imposes on mRNA export. However targeting of Sac3 which is part of a bigger complex by esa1 is a misleading statement, given that you don't show a proof of direct interactions shown, e.g. by immunoprecipiations.

    We meant to say “we tested whether Esa1 function promotes the localisation of Sac3 to the nuclear basket”. We agree that it is unknown whether this involves direct interactions between Sac3 and Esa1. We have changed the text to make this point clearer.

    13 • Page 18: "Nevertheless, our findings suggest that mammalian nucleoporins may represent a novel category of substrates for KATs and for the multiprotein complexes in which these enzymes reside, with important roles in gene expression." Given that there is little experimental evidence this statement is for my taste too strong. Rather indicate that this is a possibility which needs to be tested...

    We have changed the text as suggested.

    14 • Page 3: "Nuclear pores are macromolecular assemblies composed of approximately 30-50 different Nucleoporins": it is rather approximately 30 different nucleoporins in the species so far analyzed.

    We have corrected this as suggested.

    __Significance: __

    The concept of acetylation/deacetylation regulation of G1/S transition in budding yeast is very appealing. The specific (and important) contribution of Esa1, especially in comparison to GCN5 and Hat1 remains unclear as well as its precise effect on Nup60. Clarifying this, also in a more balanced way of presentation of discussion, would be of interest for the field.

    My research centers around NPC function.

    Audience: experts in the nuclear structure/function fields and cell cycle regulation.

    A more detailed characterisation of the specific roles of Esa1, Gcn5 and Hat1 in the G1/S transition and mRNA export will be included in a revised version, as mentioned in our response to point 3.

    __Reviewer #2 __

    Evidence, reproducibility and clarity:

    In this manuscript, Gomar-Alba et al. follow up on previous work from the lab that showed that the KDAC Hos3 is targeted to the bud neck and daughter cell nuclear pore complexes in budding yeast where it slows cell cycle progression by influencing gene positioning and nucleo-cytoplasmic transport. Overall, the current manuscript describes a well-conducted study that dissects the role of acetylation and deacetylation on Nup60 during the cell cycle using genetics and microscopy. The authors conclusively identify Esa1 as counteracting Hos3 in the nucleus (Figure 1) and show that part of their effect on cell cycle progression and gene expression is mediated by acetylation of Nup60 at K467 (Figure 2). They also demonstrate that this leads to a differential localization of several mRNA export factors and suggest that deacetylation of Nup60 blocks mRNA export in daughter cells. Although this work is overall carefully done, the last conclusion is still somewhat speculative.

    I have a number of minor suggestions to improve the manuscript, but only one major concern, which revolves around the role of chromatin tethering to NPCs. The authors have shown in their previous paper that this plays a role for CLN2 and it is known that active GAL1 interacts with the nuclear periphery, but in the current manuscript this aspect is largely disregarded although I think it could play a major role in the observed mRNA export phenotypes. Therefore, I think some additional experiments and controls as well as additional analysis are required to substantiate especially the results shown in figure 5.

    Major points:

    1. Figure 2: The authors claim that the mechanism by which Nup60 acetylation promotes cell cycle progression is the enhancement of mRNA export through the NPC. In Figure 2, the authors look at the expression levels of four candidate mRNAs which all show disturbed expression in esa1-ts which is not rescued by the nup60-KN mutation, but expression of the protein of one of these candidates (CLN2) is improved. In their previous paper, the same lab has shown that the CLN2 gene is tethered to the NPC in daughter cells with deacetylated Nup60 and that this is relieved in a Nup60 K467N mutant. I think it would be important here to investigate the protein levels of additional candidates that are not regulated at the level of gene localization. Is it a general effect that protein expression is higher in the nup60KN mutant?

    We agree this is an important point. To establish if Nup60-KN regulates only genes that interact with the NPC (such as CLN2), the reviewer suggests determining the cell cycle levels of proteins encoded by other G1/S genes that do not bind NPCs. The main problem with this approach is that with the exception of CLN2, the nuclear localisation of the (about 200) G1/S regulon genes is not yet known. In addition, establishing connections between mRNA and protein levels during the first cell cycle is only possible for short-lived proteins such as Cln2. For instance, amongst the G1/S genes shown in Figure 2, Cdc21 and Rnr1 have protein half-lives of 10 and 4 h, much longer than the 90-minute yeast cell cycle (PMID 25466257). We think a more direct approach to investigate the connection between gene position and mRNA synthesis / export would be to directly visualise the localisation of single mRNAs upon perturbation of the Nup60 acetylation pathway, using single mRNA labeling techniques (smFISH or PP7). We aim to do this for CLN2 and also for GAL1 (see point 2d of this reviewer). We will attempt these experiments for a revised version of our paper.

    1. Figure 5: In figure 5, the authors investigate the expression of a different inducible RNA (GAL1) to test whether the observed effect on mRNA export is more general. Since this is a crucial point for generalizing the finding, this data needs to be presented in a more convincing manner.

    2a. GAL1 is known to be tethered to the NPC upon transcription. Whether this tethering is affected by the Nup60-KN mutant is unclear, but since Nup60 has been implicated in GAL1 tethering in the literature, this possibility is not unlikely. GAL1 therefore becomes a similar case to CLN2, where it is difficult to disentangle effects directly due to mRNA export from the effects of gene tethering on mRNA transcription and processing. Therefore, this experiment should be repeated with a system that is independent of gene tethering. For example, induction of the GAL promoter via a b-estradiol inducible VP16 transactivator does not seem to induce tethering.

    This is an excellent idea. We are not aware of studies on the localisation of the GAL1 locus induced by a VP16 transactivator, but this was investigated for the HXK1 gene. This subtelomeric gene localises to NPCs in non-glucose carbon sources, and its localisation is perturbed by VP16 transactivation in glucose (PMID: 16760983). We will investigate whether the same is true for GAL1, and if so, perform the suggested experiments.

    2b. The activation kinetics in all mutants analyzed is very different from the wildtype. Therefore, the quantification made in Figure 5C is difficult to interpret. Therefore, it might be more fair to quantify for the mutant strains at an earlier timepoint after activation when the levels are similar to the levels in the wildtype strain. E.g. in the hos3d strain at around 250 min.

    This is a good point - indeed, persistent mother/daughter asymmetry in GAL1 expression in hos3 and nup60-KN mutants could be masked by saturated levels of GFP at late time points. An alternative way to test this is to determine the time of GAL1 induction in mother and daughter cells. We have done this in wild-type and hos3 mutant cells; our results indicate that GAL1 expression occurs first in wildt-type mothers and later in their daughters, whereas it is almost simultaneous in nup60-KN mother/daughter mutant pairs (as shown for a single M-D pair in the new figure 5A). In a revised version, we will include data of GAL1 expression for M-D pairs at different times after galactose addition for cells in figures 5C and 5E.

    2c. Similarly - although not as drastic - , in figure 5E, quantification should be done at a timepoint when the induction level is similar between DMSO and Rapamycin treated samples to make conclusions about differences between mother and daughter cell.

    We agree. See our response to the previous point.

    2d. The major claim of the paper is that mRNA export is inhibited by Nup60 deacetylation. In this figure, the mRNA levels need to be quantified to validate that it is not transcription that is affecting expression.

    We agree. In addition to regulating mRNA export (as suggested by the effect of Sac3 anchoring to NPCs) Nup60 deacetylation may also inhibit GAL1 transcription (directly, and/or indirectly via disruption of Gal1-based transcriptional feedback; PMID 23150580). To directly assess the role of Nup60 acetylation in GAL1 transcription and mRNA export, it would be ideal to determine the levels of GAL1 mRNA in both the nucleus and the cytoplasm, using smFISH and/or PP7 tools, in wild type and in mutants of the Nup60 acetylation pathway as we proposed to do for CLN2 (see our response to point 1 of this reviewer). These or equivalent experiments will be included in a revised version.

    1. The manuscript investigates in detail the effects of a KN mutant, however, a non-acetylatable mutant is not investigated. Is such a mutant viable?

    We have obtained a Nup60-KR mutant, which is predicted to behave as a non-acetylatable mimic, and it is viable. We will describe its phenotype in a revised version.

    Minor comments:

    1. Figure 2E: Is the rescue really specific to daughter cells? The dynamic range in the daughter cells is much higher due to the slower and more heterogenous timepoint of Whi5 export. However, zoom-in on the early timepoints after Whi5 import before the 30 min when 50% of the cells have exported Whi5, might reveal a significant increase of mother cells with shortened time to S phase entry. I suggest that the authors test this possibility. The cells shown in the image panels also suggest that the acetyl mimic might shorten mother cell time to S phase entry. If this is not the case, the authors might want to show a different example cell. Interestingly, it appears from the supplementary figure S5, that while Nup60 K647N partially rescues the export of Whi5, budding does not seem to be different to Nup60 wt. This appears to contradict the budding after alpha factor arrest shown in figure 2.

    We thank the reviewer for this suggestion. Indeed, zooming into the first 30 minutes shows a slight increase in the fraction of nup60-KN mother cells that export Whi5; however this change is not statistically significant when considering the entire cell population (p=0.6017, Mann-Whitney test). Therefore, we will replace the cell shown in figure 2E with a more representative example.

    As for figure S5, the reviewer is correct that in these experiments nup60-KN partially rescues Whi5 export (a marker of Start) but not budding (a downstream event), and this is indeed in variance with the experiment shown in figure 2B. Different experimental conditions may contribute to this apparent discrepancy: as noted in the text, the duration of G1 phase in cells synchronised with alpha factor is not directly comparable with that of freely cycling cells.

    1. Figure 3C: The authors use a truncated version of SAC3 for overexpression, since the full length is toxic (Figure S6A). I think it would be important to include this information in the main text.

    We agree, and have included this information in the main text.

    1. Figure 4B: Is there simply less Sac3 protein in the esa1-ts mutant? Although the authors address this question in figure S9, the very low expression levels of Sac3 may make this difficult to conclude from fluorescence quantification. A Western Blot would be an important control. The relative level of Sac3 still seems to be lower in esa1-ts daughter cells compared to mother cells, but no statistical test is shown.

    We are confident that the total Sac3-GFP levels are sufficient to make accurate comparisons, in both the nucleus and the entire cell. However, we will be happy to include western blot controls for Sac3 total levels in a revised version as the reviewer suggests. As for the levels of Sac3 in M vs D cells: Sac3 is indeed asymmetrically distributed in both wild-type and esa1-ts cells (p

    1. Analysis of mother daughter pairs (e.g. figure 5C): a paired t-test would be appropriate.

    We agree. Results do not change with this new analysis (in fact, p values are even lower for wild-type M-D pairs in figure 5C).

    1. Figure 5A: Can some representative mother-daughter pairs be shown as images for both wt and mutant in the timelapse? It is difficult to see in 5A whether there are any mother daughter pairs.

    We have modified the figure to include clearly identifiable mother-daughter pairs, as requested.

    1. Figure 4C: Please show image of localization of Sac3-GFP-FRB +/- rapamycin to the NPC.

    We have added this.

    Significance:

    This manuscript describes an important advance in understanding the role of non-histone protein modification on the regulation of cell cycle progression and gene expression. It is a logical follow-up on a previous paper from the lab (Kumar et al. 2018) and beautifully builds on this work. It is to my knowledge the first mechanistic description of regulation of nuclear pore complex function by a post-translational modification. This will therefore be a very interesting paper for anyone interested in nuclear pore complex regulation and biology, non-histone protein acetylation, asymmetric cell division, and cell cycle regulation.

    __Reviewer #3 __

    Evidence, reproducibility and clarity:

    The pre-print is dedicated to mRNA export and G1/S transition control in mother and daughter cells of budding yeasts through acetylation/deacetylation of nuclear pore component Nup60 (hsNup153). In particular, authors found that Esa1(hsTip60/KAT5) acetylates the basket nucleoporin Nup60, and this event promotes recruitment of mRNA export factors to the nuclear basket and export of polyA RNA to the cytosol. This export event promotes entry of cells into S phase; in particular, Nup60 is deacetylated by histone deacetylase Hos3 that displaces mRNA export complexes from the NPC and inhibits Start specifically in daughter cells.

    The manuscript is a well-designed and well-written study.

    Please, see my major and minor suggestions below:

    Major comments:

    P4-5. "deacetylation of the nuclear basket nucleoporin Nup60 does not affect Whi5 nuclear accumulation". I was confused by this statement because, in the previous article Kumar et al., 2018, both main text and abstract have the following phase "nuclear basket and central channel nucleoporins establish daughter-cell-specific nuclear accumulation of the transcriptional repressor Whi5.." Could you please address this discrepancy?

    Thank you for pointing this out. We should have written: “deacetylation of Nup60 does not strongly affect Whi5 nuclear accumulation”. The Kumar et al. paper shows that deacetylation of central channel nucleoporins (such as Nup49) is important to increase accumulation of Whi5 in daughter cells, whereas deacetylation of the basket nucleoporin Nup60 plays a relatively minor role (see Kumar et al, Figure 7c). We have corrected this in the main text.

    Fig.2A: In addition to increased Nup60 acetylation, I noticed an overall increased level of Nup60 after overexpression of Esa1 and Gcn5. Is it a statistically significant increase in the Nup60 level? It is not mentioned in the main text or figure legend. Does the acetylation level of Nup60 influence its stability?

    We don’t know if acetylation of Nup60 affects its stability, although it is an intriguing possibility. Although it´s true that Nup60 levels in the IP fraction seem to increase upon Esa1 and Gcn5 overexpression, nuclear levels of Nup60-mCherry are similar in wild-type, hos3∆ and nup60-KN (Supplementary Figure S11A). Therefore it is unlikely that changes in Nup60 acetylation affect its stability. We have added this information to the text.

    Authors determined the mRNA level of four representative genes in esa1-ts and esa1-ts nup60-KN cultures.

    3a. Do authors know if Nu60-KN expression affects the perinuclear positioning of these transcripts?

    We did not investigate the localisation of individual transcripts in this study. However, as mentioned in our replies to reviewer 2, we propose to do so for the CLN2 and GAL1 mRNAs, in order to test directly the effect of Nup60 acetylation in the positioning of specific mRNAs.

    3b.I also suggest authors investigate if Nup60-KN affects other transcripts using the RNAseq approach. Nup60-KN might improve the transcription output of other transcripts and it will be interesting to know if these transcripts share similar features.

    We agree that investigating the impact of Nup60 acetylation in mRNA synthesis genome-wide is an exciting challenge. We speculate that Nup60-KN is likely to have some effect in transcription, either directly or indirectly through perturbation of feedback regulatory loops caused by mRNA export defects (for instance, transcription of both CLN2 and GAL1 is regulated by positive feedback). However we think that these experiments are beyond the scope of our study, which is focused on mRNA export.

    3c. Do authors know if GAL1pr:HOS3-NLS expression affects specifically G1-dependent transcripts?

    Answering this question would require RNA sequencing experiments. As mentioned in the previous point, we think these are beyond the scope of our study. That being said, it is likely that the Hos3-Nup60 pathway downregulates gene expression during G1, because Nup60 deacetylation is largely restricted to this phase. Note that this is not the same as regulating expression of the G1/S regulon specifically, because Hos3 also regulates GAL1 expression (Figure 5). We mention this important point in the discussion (p. 17).

    3d. Another interesting question will be to define if there is a group of transcripts that respond specifically to the status of Nup60 acetylation during G1/S transition. Is it possible to make ts-driven Nup60-KN expression to turn in ON/OFF? However, this question is beyond the scope of this paper.

    Thank you for this interesting suggestion. The proposed experiment is technically possible (for example, expression of Nup60-KN could be induced in G1 using a GAL1 promoter, followed by RNA sequencing). We agree that this is beyond the scope of our paper but would like to explore the question in future studies.

    1. Fig.2D It is not mentioned that Cln2 is not cycling anymore upon Nup60-KN overexpression.

    The Cln2 protein peaks at 30 minutes in this experiment, and is degraded at approximately 120 minutes. This corresponds to the slow, incomplete G1/S transition wave of the esa1-ts nup60-KN mutant, as indicated in the budding index at the bottom of the panel. We added this in the figure 2 legend. Note that Nup60-KN is not overexpressed, since the KN mutation is inserted in the endogenous gene under the control of its native promoter.

    Fig.2E. Arrows indicating Whi5 export timing do not match to the numbers in the main text. For example, yellow arrows indicate Whi5 export in wt strain at 30 and 78 min, but it is stated 15 and 59 min in the text. Also, do I understand right that Whi5-mCherry is not visible in the cytosol?

    See our reply to reviewer 2, point 4: we will replace the cell shown in figure 2E with a more representative example. As for Whi5-mCherry, it is visible in the cytoplasm but only weakly (since it is diluted into the larger cytoplasmic volume), and not at all in the images shown due to the overlay with the brightfield channel.

    Did the authors analyze where SAC3 and MTR2 are localized in hos3del, Nup60KN, and Esa-ts strains once their localization was affected in the nucleus? Is the overall level Sac3 level is affected in hos3del and Nup60KN strains?

    We have imaged the localisation of Sac3-GFP and Mtr2-GFP during the whole cycle using time-lapse microscopy. Our impression is that in wild type cells, their perinuclear levels increase during S phase in daughter cells, which mirrors the increase in Nup60 acetylation. In contrast, Sac3 and Mtr2 perinuclear levels seem more stable in hos3 and nup60-KN cells. We will include these analyses in a revised version. The total level of Sac3 is not affected, as shown in the updated figure 4; see our reply to reviewer 2, point 6.

    Fig4C. "Sac3-GFP-FRB partitioned equally to M and D nuclei, in the presence of Nup60-mCherry-FKBP and rapamycin (Figure 4C)." Sac3-GFP-FRB is slightly elevated in mother cells. Did you run a statistical test between the first and the third column on the box plot?

    Comparing the first and third columns in Fig 4C (Nup60 and Sac3 in control cells) shows that the mother cell accumulation is higher for Sac3 than for Nup60 (p

    P15. "GAL1 expression levels were higher in wild-type mother cells than in their daughter, and these differences were absent in cells lacking Hos3 or expressing Nup60KN". GAL1-10 promoter contains information necessary and sufficient for recruitment to the nuclear periphery (PMID: 27489341). I wonder if GAL1pr-driven transgenes of HOS3, spt10, hat1, and etc., contain DNA sequences sufficient for targeting genes to the nuclear periphery, and these genes are asymmetrically expressed in mother and daughter cells because of the presence of GAL1pr?

    We agree that these genes may be expressed at different levels in mother and daughter cells. We don’t think this asymmetric expression affects our conclusions. Indeed, the phenotypes scored (growth on plates) apply to the population and not to individual cells. The one exception is figure 3D, in which mRNA nuclear accumulation is scored in single cells. In this case, it remains possible that some of the variability observed corresponds to differences between mothers and daughters. In this case, our measurements could under-estimate the effect of Hos3-NLS in inhibition of mRNA export. However, since we cannot differentiate M and D cells in this experiment, we prefer not to speculate on this possibility in the text.

    Minor comments:

    Supplementary Fig. S1, it will be easy to read cell viability assays if 1A, S1A and S1B figures have the same orientation.

    We have changed the figure as suggested.

    Could you please clarify the difference between HOS3-NLS and GAL1pr:HOS3-NLS in the text of figure legend? P.33

    We have fixed this (figure 1 legend).

    P6. I recommend adding the following sentence to help clarity of the text: "To understand how NPC acetylation regulates the G1/S transition (Start), we sought to identify the lysine acetyl-transferases (KATs) counteracting the activity of the Hos3 deacetylase. Hos3 displays asymmetric distribution between mother and daughter cells in wild type Saccharomyces cerevisiae. Overexpression of a version of Hos3 fused to a nuclear localization signal (GAL1pr-HOS3-NLS) leads to targeting of Hos3 to mother and daughter cell nuclei, deacetylation of nucleoporins, and inhibition of cell proliferation (Kumar et al, 2018)."

    We thank the reviewer for this suggestion. This has been added.

    P8. Misspelling: Though Nup60 acetylation

    This has been fixed.

    FigS7. Description of polyA distribution is missing for single gcn5del strain.

    Thank you for pointing this out. This has been added.

    Misspelling: We conclude that Esa1 and Nup60 acetylation promotes Start, at least in part, by targeting Sac3 to the nuclear basket, where it mediates mRNA export.

    This has been fixed.

    Significance

    Authors of this pre-print overview and try to resolve a fundamental and not well-studied question about NPC acetylation status and S phase entry. This work is a logical extension of their previously published work (PMID: 29531309). However, this study for the first-time links status of NPC acetylation to mRNA export through lysine acetyl transferases. It will be interesting to address this question in mammalian cells considering interaction of basket nucleoporins with Tip60/KAT5 (PMID: 24302573).

    This work might be of interest to researchers investigating RNA export, transcription regulation, and nuclear pores.

    My fields of expertise are RNA export, nucleoporins, transcription regulation.

    I do not have expertise to evaluate yeast strains used in this study.

  3. Review Commons

    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 pre-print is dedicated to mRNA export and G1/S transition control in mother and daughter cells of budding yeasts through acetylation/deacetylation of nuclear pore component Nup60 (hsNup153). In particular, authors found that Esa1(hsTip60/KAT5) acetylates the basket nucleoporin Nup60, and this event promotes recruitment of mRNA export factors to the nuclear basket and export of polyA RNA to the cytosol. This export event promotes entry of cells into S phase; in particular, Nup60 is deacetylated by histone deacetylase Hos3 that displaces mRNA export complexes from the NPC and inhibits Start specifically in daughter cells.

    The manuscript is a well-designed and well-written study.

    Please, see my major and minor suggestions below:

    Major comments:

    1. P4-5. "deacetylation of the nuclear basket nucleoporin Nup60 does not affect Whi5 nuclear accumulation". I was confused by this statement because, in the previous article Kumar et al., 2018, both main text and abstract have the following phase "nuclear basket and central channel nucleoporins establish daughter-cell-specific nuclear accumulation of the transcriptional repressor Whi5.." Could you please address this discrepancy?
    2. Fig.2A: In addition to increased Nup60 acetylation, I noticed an overall increased level of Nup60 after overexpression of Esa1 and Gcn5. Is it a statistically significant increase in the Nup60 level? It is not mentioned in the main text or figure legend. Does the acetylation level of Nup60 influence its stability?
    3. Authors determined the mRNA level of four representative genes in esa1-ts and esa1-ts nup60-KN cultures. Do authors know if Nu60-KN expression affects the perinuclear positioning of these transcripts? I also suggest authors investigate if Nup60-KN affects other transcripts using the RNAseq approach. Nup60-KN might improve the transcription output of other transcripts and it will be interesting to know if these transcripts share similar features. Do authors know if GAL1pr:HOS3-NLS expression affects specifically G1-dependent transcripts?

    Another interesting question will be to define if there is a group of transcripts that respond specifically to the status of Nup60 acetylation during G1/S transition. Is it possible to make ts-driven Nup60-KN expression to turn in ON/OFF? However, this question is beyond the scope of this paper.

    1. Fig.2D It is not mentioned that Cln2 is not cycling anymore upon Nup60-KN overexpression.
    2. Fig.2E. Arrows indicating Whi5 export timing do not match to the numbers in the main text. For example, yellow arrows indicate Whi5 export in wt strain at 30 and 78 min, but it is stated 15 and 59 min in the text. Also, do I understand right that Whi5-mCherry is not visible in the cytosol?
    3. Did the authors analyze where SAC3 and MTR2 are localized in hos3del, Nup60KN, and Esa-ts strains once their localization was affected in the nucleus? Is the overall level Sac3 level is affected in hos3del and Nup60KN strains?
    4. Fig4C. "Sac3-GFP-FRB partitioned equally to M and D nuclei, in the presence of Nup60-mCherry-FKBP and rapamycin (Figure 4C)." Sac3-GFP-FRB is slightly elevated in mother cells. Did you run a statistical test between the first and the third column on the box plot?
    5. P15. "GAL1 expression levels were higher in wild-type mother cells than in their daughter, and these differences were absent in cells lacking Hos3 or expressing Nup60KN". GAL1-10 promoter contains information necessary and sufficient for recruitment to the nuclear periphery (PMID: 27489341). I wonder if GAL1pr-driven transgenes of HOS3, spt10, hat1, and etc., contain DNA sequences sufficient for targeting genes to the nuclear periphery, and these genes are asymmetrically expressed in mother and daughter cells because of the presence of GAL1pr?

    Minor comments:

    1. Supplementary Fig. S1, it will be easy to read cell viability assays if 1A, S1A and S1B figures have the same orientation.
    2. Could you please clarify the difference between HOS3-NLS and GAL1pr:HOS3-NLS in the text of figure legend? P.33
    3. P6. I recommend adding the following sentence to help clarity of the text: "To understand how NPC acetylation regulates the G1/S transition (Start), we sought to identify the lysine acetyl-transferases (KATs) counteracting the activity of the Hos3 deacetylase. Hos3 displays asymmetric distribution between mother and daughter cells in wild type Saccharomyces cerevisiae. Overexpression of a version of Hos3 fused to a nuclear localization signal (GAL1pr-HOS3-NLS) leads to targeting of Hos3 to mother and daughter cell nuclei, deacetylation of nucleoporins, and inhibition of cell proliferation (Kumar et al, 2018)."
    4. P8. Misspelling: Though Nup60 acetylation
    5. FigS7. Description of polyA distribution is missing for single gcn5del strain.
    6. Misspelling: We conclude that Esa1 and Nup60 acetylation promotes Start, at least in part, by targeting Sac3 to the nuclear basket, where it mediates mRNA export.

    Significance

    Authors of this pre-print overview and try to resolve a fundamental and not well-studied question about NPC acetylation status and S phase entry. This work is a logical extension of their previously published work (PMID: 29531309). However, this study for the first-time links status of NPC acetylation to mRNA export through lysine acetyl transferases. It will be interesting to address this question in mammalian cells considering interaction of basket nucleoporins with Tip60/KAT5 (PMID: 24302573).

    This work might be of interest to researchers investigating RNA export, transcription regulation, and nuclear pores.

    My fields of expertise are RNA export, nucleoporins, transcription regulation.

    I do not have expertise to evaluate yeast strains used in this study.

  4. Review Commons

    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 this manuscript, Gomar-Alba et al. follow up on previous work from the lab that showed that the KDAC Hos3 is targeted to the bud neck and daughter cell nuclear pore complexes in budding yeast where it slows cell cycle progression by influencing gene positioning and nucleo-cytoplasmic transport. Overall, the current manuscript describes a well-conducted study that dissects the role of acetylation and deacetylation on Nup60 during the cell cycle using genetics and microscopy. The authors conclusively identify Esa1 as counteracting Hos3 in the nucleus (Figure 1) and show that part of their effect on cell cycle progression and gene expression is mediated by acetylation of Nup60 at K467 (Figure 2). They also demonstrate that this leads to a differential localization of several mRNA export factors and suggest that deacetylation of Nup60 blocks mRNA export in daughter cells. Although this work is overall carefully done, the last conclusion is still somewhat speculative.

    I have a number of minor suggestions to improve the manuscript, but only one major concern, which revolves around the role of chromatin tethering to NPCs. The authors have shown in their previous paper that this plays a role for CLN2 and it is known that active GAL1 interacts with the nuclear periphery, but in the current manuscript this aspect is largely disregarded although I think it could play a major role in the observed mRNA export phenotypes. Therefore, I think some additional experiments and controls as well as additional analysis are required to substantiate especially the results shown in figure 5.

    Major points:

    1. Figure 2: The authors claim that the mechanism by which Nup60 acetylation promotes cell cycle progression is the enhancement of mRNA export through the NPC. In Figure 2, the authors look at the expression levels of four candidate mRNAs which all show disturbed expression in esa1-ts which is not rescued by the nup60-KN mutation, but expression of the protein of one of these candidates (CLN2) is improved. In their previous paper, the same lab has shown that the CLN2 gene is tethered to the NPC in daughter cells with deacetylated Nup60 and that this is relieved in a Nup60 K467N mutant. I think it would be important here to investigate the protein levels of additional candidates that are not regulated at the level of gene localization. Is it a general effect that protein expression is higher in the nup60KN mutant?

    2. Figure 5: In figure 5, the authors investigate the expression of a different inducible RNA (GAL1) to test whether the observed effect on mRNA export is more general. Since this is a crucial point for generalizing the finding, this data needs to be presented in a more convincing manner.

    a. GAL1 is known to be tethered to the NPC upon transcription. Whether this tethering is affected by the Nup60-KN mutant is unclear, but since Nup60 has been implicated in GAL1 tethering in the literature, this possibility is not unlikely. GAL1 therefore becomes a similar case to CLN2, where it is difficult to disentangle effects directly due to mRNA export from the effects of gene tethering on mRNA transcription and processing. Therefore, this experiment should be repeated with a system that is independent of gene tethering. For example, induction of the GAL promoter via a b-estradiol inducible VP16 transactivator does not seem to induce tethering.

    b. The activation kinetics in all mutants analyzed is very different from the wildtype. Therefore, the quantification made in Figure 5C is difficult to interpret. Therefore, it might be more fair to quantify for the mutant strains at an earlier timepoint after activation when the levels are similar to the levels in the wildtype strain. E.g. in the hos3d strain at around 250 min.

    c. Similarly - although not as drastic - , in figure 5E, quantification should be done at a timepoint when the induction level is similar between DMSO and Rapamycin treated samples to make conclusions about differences between mother and daughter cell.

    d. The major claim of the paper is that mRNA export is inhibited by Nup60 deacetylation. In this figure, the mRNA levels need to be quantified to validate that it is not transcription that is affecting expression.

    1. The manuscript investigates in detail the effects of a KN mutant, however, a non-acetylatable mutant is not investigated. Is such a mutant viable?

    Minor comments:

    1. Figure 2E: Is the rescue really specific to daughter cells? The dynamic range in the daughter cells is much higher due to the slower and more heterogenous timepoint of Whi5 export. However, zoom-in on the early timepoints after Whi5 import before the 30 min when 50% of the cells have exported Whi5, might reveal a significant increase of mother cells with shortened time to S phase entry. I suggest that the authors test this possibility. The cells shown in the image panels also suggest that the acetyl mimic might shorten mother cell time to S phase entry. If this is not the case, the authors might want to show a different example cell. Interestingly, it appears from the supplementary figure S5, that while Nup60 K647N partially rescues the export of Whi5, budding does not seem to be different to Nup60 wt. This appears to contradict the budding after alpha factor arrest shown in figure 2.

    2. Figure 3C: The authors use a truncated version of SAC3 for overexpression, since the full length is toxic (Figure S6A). I think it would be important to include this information in the main text.

    3. Figure 4B: Is there simply less Sac3 protein in the esa1-ts mutant? Although the authors address this question in figure S9, the very low expression levels of Sac3 may make this difficult to conclude from fluorescence quantification. A Western Blot would be an important control. The relative level of Sac3 still seems to be lower in esa1-ts daughter cells compared to mother cells, but no statistical test is shown.

    4. Analysis of mother daughter pairs (e.g. figure 5C): a paired t-test would be appropriate.

    5. Figure 5A: Can some representative mother-daughter pairs be shown as images for both wt and mutant in the timelapse? It is difficult to see in 5A whether there are any mother daughter pairs.

    6. Figure 4C: Please show image of localization of Sac3-GFP-FRB +/- rapamycin to the NPC.

    Significance

    This manuscript describes an important advance in understanding the role of non-histone protein modification on the regulation of cell cycle progression and gene expression. It is a logical follow-up on a previous paper from the lab (Kumar et al. 2018) and beautifully builds on this work. It is to my knowledge the first mechanistic description of regulation of nuclear pore complex function by a post-translational modification. This will therefore be a very interesting paper for anyone interested in nuclear pore complex regulation and biology, non-histone protein acetylation, asymmetric cell division, and cell cycle regulation.

  5. Review Commons

    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:

    Acetylation/Deacetylation controls G1/s transition in budding yeast. The lysine acetyl transferase Esa1 is here shown to play a role, in part via acetylation of the nuclear pore complex basket component Nup60, which stimulates mRNA export.

    Major comments:

    • Figure 1C: The curve for esa1-ts in this figure and the curve in the supplementary figure S2B are not similar, while the first shows 10% cells budding after 60 minutes it is about 50% after 60 min in S2B. Another helpful way of presenting the data could be the length of the G1 phase (from cytokinesis to budding) in the WT, esa1-ts, gcn5delta cells over time.

    • What is the rational of creating the Nup60-KN mutation. Does it prevent acetylation of Nup60, at least by GCN5 and/or esa1?

    • Given the much stronger phenotype of the esa1-ts+GCN5 delta condition for G1/S transition as compared to esa1-ts and that GCN5 seems to strongly acetylate Nup60 I do not understand the sole focus on esa1 in the study. The fact that the Nup60-KN cells do not show G1/S transition under esa1-ts+GCN5 delta conditions in experiments presented in Fig. S3 argues that esa1 meaidted acetylation of Nup60 is only one, probably minor aspect of G1/S transition. This should be much balanced discussed.

    • Suppl: Fig 2: I miss the hat1delta+gcn5delta condition.

    Minor comments:

    • Figure legend 2C "at least 200 cells were scored": please state number of replicates

    • Figure 2E: X axis "impor" should be corrected to "import"

    • Would Mex67 and/or Mrt2 overexpression recue the esa1-ts and esa1-ts+GCN5 delta phenotype?

    • Figure 4 A: The size of the daughter cells in the hos3delta condition seems smaller as compared to esa1-ts. Is this true and can you comment this? Is a premature onset of S phase observed here?

    • Figure 4D: The still images in figure 2E and 4D do not correspond with the quantitation. E.g. in Fig 2E the esa1ts cells shows Whi5 export at t=81 min, which is according to the shown quantitation unusual late.

    • Figure 4B: it is not clear why for the quantitation a different representation is chosen as compared to 4A. It would be better to show the nuclear intensities of mother/daughter as in Figure 4A.

    • Figure 4D: To strengthen these results, it would be good to perform this assay with esa1-ts Nup60-KN cells as in figure 2a. The release of Whi5-GFP is expected to behave in a similar way to the WT. This would ensure that Nup60 acetylation is a pre-requisite for Whi5 release

    • Page 13 "Finally, we tested whether Esa1 targets Sac3 to G1 nuclei": The effect of esa1 knockdown on Sac3 fit with the story line and the effect esa1 imposes on mRNA export. However targeting of Sac3 which is part of a bigger complex by esa1 is a misleading statement, given that you don't show a proof of direct interactions shown, e.g. by immunoprecipiations.

    • Page 18: "Nevertheless, our findings suggest that mammalian nucleoporins may represent a novel category of substrates for KATs and for the multiprotein complexes in which these enzymes reside, with important roles in gene expression." Given that there is little experimental evidence this statement is for my taste too strong. Rather indicate that this is a possibility which needs to be tested...

    • Page 3: "Nuclear pores are macromolecular assemblies composed of approximately 30-50 different

    • Nucleoporins": it is rather approximately 30 different nucleoporins in the species so far analyzed.

    Significance

    The concept of acetylation/deacetylation regulation of G1/S transition in budding yeast is very appealing. The specific (and important) contribution of Esa1, especially in comparison to GCN5 and Hat1 remains unclear as well as its precise effect on Nup60. Clarifying this, also in a more balanced way of presentation of discussion, would be of interest for the field.

    My research centers around NPC function.

    Audience: experts in the nuclear structure/function fields and cell cycle regulation.

  6. Version published to 10.1101/2021.09.01.458533 on bioRxiv