Differential nuclear import regulates nuclear RNA inheritance following mitosis

  1. Michael D. Blower
  2. Wei Wang
  3. Judith A. Sharp

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Abstract

The authors investigate inheritance of nuclear RNA by G1 cells following mitosis. They find that multiple pathways regulate differential inheritance of nuclear RNAs.

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  1. Version published to 10.1091/mbc.e23-01-0004
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    Reply to the reviewers

    Reviewer 1.

    Major point:

    (1) The authors rely upon the redistribution of RNA to measure the inheritance of extant RNAs following cell cycle release. Blocking transcription nicely shows new synthesis is not required for this inheritance. This is also consistent with the idea any newly synthesized RNA would be 'dark,' or not EU labeled, but the transcription inhibitor experiments are critical controls and nicely done. As hinted at the end of their discussion, however, a lack of RNA localizing to G1 chromosomes could be formally attributable to differential RNA stability. Might altered RNA stability of NEAT1, MALAT1, or U2 also contribute to the observed altered localizations upon interphase reentry? The authors could use qPCR or measure RNA half-life to test this possibility. These data would nicely compliment the authors' existing FISH experiments and allow them to specifically argue for differential RNA localization.

    We have addressed this point by measuring the stability of MALAT1, U2, and NEAT1 in G2 cells after transcription inhibition using RNA FISH. We find that U2 and MALAT1 exhibit very little RNA degradation after 2.5 hours of transcription inhibition, which is consistent with the reported half-lives for each of these transcripts (10 hours for MALAT1 and >24hrs for U2; PMC3337439). We conclude that differential RNA stability cannot account for differential RNA import observed for these two transcripts. In contrast, NEAT1 transcript is almost undetectable after 1.5 hours of transcription inhibition, which is also consistent with the reported half-life of this transcript (22406755, 3337439). Therefore, RNA degradation during mitosis could contribute to a lack of NEAT1 nuclear import in G1. We have included this new data in a modified Figure 2E (text p5 lines 154-166).

    Minor Points:

    (1) The authors examine published datasets identifying RNA associated with chromatin and state the reason why these data show little overlap is "primarily attributable to purification methodology." This statement seems speculative, and its basis seems unclear.

    We have changed the wording of this section to remove unwarranted speculation (p4-5 lines 116-129).

    (2) The SAF-A-AA experiments failed to reveal insight into mechanisms of RNA sorting, although they do suggest the AA construct functions as a gain-of-function due to a) increased RNA reincorporated into chromosomes b) dramatic increase of chromosome targeting of SAF-A. These effects make it difficult to interpret the SAF-A-AA data. Related to this point, the analysis of altered RNA distributions relative to SAF-A is underdeveloped. Because the authors only examined one lncRNA (MALAT1), the conclusion that “forced retention of SAF-A on mitotic chromatin does not lead to an increase in the nuclear inheritance of specific transcripts” seems like an overstatement.

    We have reworded this conclusion about the role of SAF-A-AA on mitotic chromatin retention to more accurately reflect our findings (p6 line 197). (3) The authors find the U2 spliceosomal RNA is preferentially inherited. Might they speculate why this would be advantageous?

    We have added a sentence to the discussion speculating about the importance of U2 inheritance (p8 line 269-271). (4) Optional: it would be exciting to test the significance of U2 RNA inheritance

    We agree with the reviewer that this would be an exciting future direction to test. We envision that testing this idea rigorously would require the development of several new degron cell lines and is outside the scope of this study. (5) For Figure 1, please add statistics to figures and legend; add N=cells examined.

    We have added a new supplemental Excel spreadsheet that contains the N of cells measured for each experiment and added statistics to figure legends and figures where tests were significant. (6) For Figure 2, single channel panel of U2 RNA should be added. Figure 2E seems to reproduce the same data shown in Figure 2D (right-most columns) shown with different axes.

    We have added a single channel image of U2 to Figure 2 and replaced panel 2E with analysis of MALAT1, NEAT1, and U2 stability after transcription inhibition. (7) Figure 3, it is unclear why the authors selected MALAT1 for analysis, but not NEAT1 (or the single (unlabeled) antisense RNA also enriched in the SAF-A IP (figure 2C).

    We examined MALAT1 in greater detail because it is the most abundant lncRNA bound by SAF-A and most robust RNA FISH probe. The unlabeled antisense transcript is hnRNPUas1 and was not detectable in DLD1 cells by RNA FISH. (8) Figure 4B, please add statistics to figure and legend.

    For this experiment we prefer not to add statistics to the figure. This experiment was performed on a limited number of cells (21 and 8 respectively) and we do not believe that it is statistically appropriate to treat each cell as an independent N. The data confirms results in our previously published work (Sharp et al 2020) using live cell imaging. (9) Methods: in their description of the published lists of chromatin-bound RNAs, the authors should cite those works and provide a data availability statement with the associated GEO

    We have cited these works in the text and methods sections and added GEO accession numbers associated with these studies. (p21 line 442).

    Reviewer 2

    Major comments:

    The authors pose an interesting question -- how does nuclear RNA segregate following mitosis. In many ways, the results presented in this manuscript are rather preliminary. Key controls and validation are missing. Because of this, it is difficult to assess the validity of the main conclusions of the study. More specifically:

    1. The main conclusion of the manuscript ("about half of nuclear RNA is inherited by G1 cells following division") is primarily dependent on the experiment described in Fig 1A-B. The authors labeled synchronized cells with EU and quantified nuclear signal after release from synchronization. However, key controls are missing. What is the synchronization efficiency of the RO3306 treatment? How many cells in their acquired fields of cells are in G2 vs in other cell cycle stages? Following their drug release, what percentage of the synchronized cells have undergone telophase? What is the potential error rate in identifying the cell cycle stage using their visual imaging analysis? Without these key controls, it is unclear how to interpret the data presented in Fig 1B.

    One reason that nuclear inheritance has not been properly addressed in the literature is the difficulty in obtaining pure populations of cells synchronized in telophase or recently divided cells in early G1. There are no drugs available which can uniquely target these cell stages. In addition, the ability of human cells to all release perfectly synchronously from a drug-induced arrest can vary with cell type. For this reason we used a strategy employing synchronization methods designed to enrich cell populations for telophase or early G1 events, combined with single cell analysis of events with the distinct cytological features of each stage. Cells that have recently divided are extremely distinctive and easily identified using a combination of DAPI morphology to assess nuclear size and condensation state and the presence of Aurora-B/Midbody staining to indicate a recent cytokinesis. Our approach of using single cell analysis coupled with quantitative imaging therefore does not require a high efficiency of synchronization in cell populations. To gain confidence that our observations were reproducible we analyzed a large number of cells, performed multiple experimental replicates, and applied statistical tests to the data.

    To clarify these important points we have added text to the descriptions of how these experiments were performed (p3 line 72) and added information about the number of biological replicates to all figure legends and number of cell analyzed in each experiment to Supplementary Table 1.

    1. The use of transcriptional inhibitors in Fig 1 is really nice and is important for showing that it's not due to new transcription following mitosis. Well done!
    1. One potential mechanism that could explain the observed 25% relocalized nuclear RNA is through passive diffusion. That is, a proportion of molecules that are randomly diffusing during mitosis get trapped inside the newly formed nuclear membrane in early G1. This would be considered noise, and not a specific process that actively relocalizes nuclear RNA back into the nucleus. However, the authors' assay does not have a measure of the noise in their system. One potential experiment that may help quantify this noise is to express GFP in their cells, perform the experiment described in Fig 1A, and quantify the nuclear signal after telophase. This quantification would be the lower bound of the random process. A similar experiment with GFP-NLS could be performed to assess the upper bound of the 'inherited' molecules after mitosis. Without this type of control to quantify noise/random diffusion levels, it is unclear how much of the 25% EU signal that the authors detect is specific to the process they are testing.

    We appreciate the point that the reviewer has raised. To address this concern we examined the localization of the abundant mRNA b-actin. We examined the fraction of all b-actin FISH signal that is present in the nucleus in G2 and G1 cells following division. If a significant fraction of RNA is trapped in the reforming nucleus then we would have expected the fraction of b-actin in the G1 nucleus to increase. We observed that less b-actin RNA was present in the G1 nucleus, suggesting that passive entrapment of RNA is unlikely to be a mechanism of RNA inheritance. This is consistent with a lack of inheritance of MALAT1 and NEAT1 lncRNAs following mitosis. We have added these results to a new Supplemental Figure 2 and added text describing the results to the Results section of the manuscript (p4 lines 101-113). Additionally, this result is consistent with recent work showing that mitotic chromosomes condense through histone deacetylation and exclude negatively charged macromolecules (PMID: 35922507) and that chromosome clustering by Ki67 in early G1 phase excludes the cytoplasm from the new nucleus (PMID: 32879492). These references and ideas are now included in the results section of the manuscript.

    Related to the comment 1 and 2, EU labeling for 3 hrs in G2 cells would label ALL transcribed RNA, which would include mature mRNAs that will be translated in the cytoplasm. That is, this method is not specific to labeling nuclear RNAs only. How much of their signal is from mRNAs that got trapped inside the newly formed nuclear membrane? One way to test this is to measure the nuclear EU signal at later time points following telophase. Presumably, the nuclear transport mechanism would lead to export of non-nuclear RNAs and only the retained nuclear RNAs would contribute to the signal.

    Please see our response to point 3 with regard to entrapment. The laboratory that originally described EU RNA labeling demonstrated a 3 hour EU labeling period results in labeling nuclear RNA, and that longer labeling periods are required to visualize EU labeling of cytoplasmic RNAs after export (18840688). We have also observed in our previously published work that the 3 hour period labels nuclear RNA during interphase (33053167, 32035037). The nuclear EU signal reflects RNAs undergoing transcription, nuclear retained RNAs, and mature mRNAs prior to nuclear export.

    To identify nuclear RNAs that could be relocalized following mitosis, the authors analyzed data from "two different studies using different methodologies and a total of three different cell lines". From this analysis, the authors "found very little overlap in the chromatin-bound RNAs identified in these studies (Fig 2A)". This analysis seems fraught with problems. What is the rationale for using these studies? How valid is it to compare results from different methodologies and from different cell lines from the DLD-1 cells used in this study?

    We analyzed the data from these two studies because they were the only published studies that identified RNAs that were tightly linked to chromatin. We chose to compare the results from three different human cell lines because we sought to identify nuclear RNAs that were cell type-independent, so that we could analyze the transcripts behavior in DLD1 cells. In support of using these two studies all the RNAs that we analyzed were nuclear in our RNA FISH assays.

    A known problem of assessing chromatin-bound RNAs is that the level of contamination from cytoplasmic RNAs is highly variable and highly dependent on the assay. Indeed some of the most common contaminants of nuclear RNA assays are sn-, and sno-RNAs, and these are the main classes of RNA that the authors identified as common among the three data sets. What validation was used to assess whether these are the common noise/contaminants in the data?

    Our goal in using the two previously published studies was to identify cell type-independent nuclear RNAs that could be studied in detail using FISH. For validation in our study we performed RNA FISH on MALAT1, NEAT1, and U2. We found that each of these RNAs are highly enriched in the nucleus, consistent with previous publications. Since snRNAs function in splicing and snoRNA primarily function in the modification of tRNA and rRNA in the nucleolus it seems unlikely that these are contaminants of nuclear preparations. Each of the published studies performed their own validations of their purification and sequencing methodology. For the purpose of our work nuclear enrichment of a transcript by RNA FISH satisfied our requirements.

    One experimental validation that can be performed is biochemical fractionation of EU labeled cells, which would allow for fractionating nuclear from cytoplasmic RNA. The same problems arise with the analysis shown in Fig 3C when comparing SAF-A RIP-seq with this merged list of chromatin bound RNAs.

    In support of the nuclear enrichment of each of the transcripts that we examined RNA-FISH analysis demonstrated significant nuclear enrichment. Additionally, many previous studies have shown that each of these transcripts are enriched in the nucleus (U2: 11489914, 10021385, 7597053; NEAT1: 17270048; MALAT1: 12970751, 17270048). New text describing our use of these studies is present in the results section (p4-5 lines 117-129).

    Throughout the manuscript, the authors pose their findings as "RNA inheritance" following mitosis. However, this terminology is misleading. In fact, unless RNAs are lost/kicked out of the cell as they divide, aren't all RNAs inherited following cell division since they are present in the new daughter cells? Instead, what the authors mean is that some nuclear RNAs retain their function following cell division by relocalizing back into the nucleus in the new G1 cells, whereas other nuclear RNAs are unable to relocalize into the nucleus, and then presumably turned over by degradation process. The authors should take better care of their terminology throughout the manuscript.

    Thank you for pointing this out to us. As the reviewer stated most nuclear RNAs are removed from chromatin during mitosis. Only a subset are reimported into the nucleus. We have modified our wording to clearly state that we are discussing nuclear RNA inheritance by daughter cell nuclei rather than inheritance into daughter cells in general. These text changes can be found throughout the manuscript.

    Minor comments:

    1. In all of the figures showing quantification of nuclear EU/FISH signal, the colors (red v blue) are not described (not found in the legend or methods). Presumably they are biological replicates, but this should be clearly stated.

    We have modified the plots and figure legends to more clearly explain what is plotted (See text in Figure Legends).

    1. Is figure 2E the same data presented in Fig 2D but in different y-axis? If so, state clearly

    We have removed the data in the previous version of Figure 2E and replaced it with new data examining stability of MALAT1, NEAT1, and U2 in response to Reviewer 1 (p5 lines 154-166).

    Figure 3A. This experiment is using the SAF-A-AID-mCherry system. Therefore the label in Fig 3A should be SAF-A-KD (Knockdown) instead of KO (knockout)

    We have corrected this in Figure 3.

    1. Typo in Fig 4B y-axis. It should be "Chromatin-localized SAF-A" instead of "Chromain-localized SAF-A"

    Thank you for pointing this out, we have corrected it.

    1. The methods section indicate the "precise N or replicates in indicated figure legends" but none of the figure legends have the N values listed.

    We have listed number of biological replicates in all figure legends and included a new Supplemental Table 1 that contains the number of cells measured for each experiment.

    Reviewer 3

    The authors investigate an interesting question focussed on whether nuclear RNA from the previous cell cycle is present in the subsequent G1. It turns out that this is more complex than expected with some classes of RNA being inherited whilst others are not. SAF-A or HNRNPU had been implicated in this process but the authors suggest that its role is limited.

    Figure 1 In panel A the authors write on image SAF-A-mCh. What does this refer to?

    We have added information to the Figure legends indicating that this refers to SAF-A-AID-mCherry knocked-in to the endogenous SAF-A locus (see Figure Legends).

    Panel B and other panels can the authors present this data as a boxplot or distribution plot to get a better feel of the data distribution spread.

    We have modified all the plots in the manuscript to the Superviolin form to provide a clearer depiction of experimental replicates, mean, and standard deviation.

    Presumably labelled RNAs are naturally turned over. Have the authors considered that some loss of signal could be because of this?

    We have addressed the stability of specific RNAs using RNA FISH. We find that U2 and MALAT1 show essentially no degradation during the time course of our experiments. This data has now been included in an updated Figure 2. We have also modified our text to address this point more clearly (Figure 2E and p5 lines 154-166).

    Panel E, have the authors considered labelling RNA before RO3306 treatment? What effect would this have?

    We have performed this experiment in RPE1 cells and the presence of RO3306 did not affect cytological detection of transcript labeling. We have not included this experiment in the manuscript because it is performed in a different cell line than we use for the remainder of these studies.

    Shouls TI be added before RO3306 washout?

    We added transcription inhibitors after RO washout and entry into mitosis because transcription is naturally suppressed during mitosis. We were concerned that transcriptional inhibition in late G2 could lead to failure to properly enter into M phase.

    Also, it is unclear what the arrows are pointing at. In panel F there is a difference between the red and blue experiments. In the methods the authors say that inhibition was for either 1.5 or 2 h. Is this the source of the difference?

    We have modified the figure legends to state clearly that different colors indicate biological replicate experiments (See Figure Legends). Figure 2 In panel A there are clear differences between the cell lines. Is it right to compare them? Particularly the GRID-seq vs diMARGI? B, how relevant is it focussing on the "42" overlapping RNAs? In my mind this is not very informative.

    Our goal with this analysis was to identify cell type-independent chromatin bound RNAs to analyze in greater detail. Therefore, we analyzed three different cell lines because we planned to analyze transcript behavior in DLD1 cells, which were not included in either study. We have explained this rationale in greater detail in a revised version of the text (p4-5 lines 116-129).

    D-E, at a glance it is not clear that E is an expanded view of D. It might be easier if the panels were at same height.

    We have removed Panel E and replaced it with a new experiment examining the stability of NEAT1, MALAT1, and U2 after transcription inhibition (p5 lines 154-166). Figure 3 Is it correct to describe IAA treated degron cells as a KO? I also could not see a WB showing how complete SAF-A KD was.

    We previously characterized these cell lines in great detail (Sharp et al. JCB 2020). We have now provided quantitative measurement of SAF-A-mCherry fluorescence after different times of auxin addition to provide a quantitative estimate of SAF-A depletion (Supplemental Figure 3C).

    2 h treatment seems quite short, is this enough time to obtain sufficient knock down? How heterogenous is SAF-A KD in the cell population?

    We examined SAF-A depletion by auxin addition at 2 hours and 24 hours and achieve comparable depletion levels. This data in now included in Supplementary Figure 3C. There is some heterogeneity in the KD as is evident in Figure S3C, but these cells are easily identifiable by the presence of SAF-A-AID-mCherry fluorescence.

    Previous studies have shown that SAF-A does not like being tagged. How certain are the authors that these cells behave typically?

    We have generated two different cell lines (DLD1 and RPE1) where a C-terminal tag is inserted into the both copies of the endogenous SAF-A gene. SAF-A is one of the common essential genes (https://depmap.org/portal/gene/HNRNPU?tab=overview), however each of our cell lines exhibits no growth defects. We have recently shown that C-terminally tagged SAF-A fully rescues SAF-A knockout phenotypes (Sharp et al. JCB. 2020). Additionally, we have also performed RNA-seq (not published) on RPE1, RPE1 with endogenously tagged SAF-A and RPE-1 depleted of SAF-A and rescued with WT SAF-A-GFP and observed no changes in gene expression or mRNA splicing. Based on these assays we are confident that C-terminally tagged SAF-A expressed at endogenous levels functions normally. Figure 4 I'm struggling with the heading, and wonder if this is not supported by the data. Similarly the final sentence "The highly dynamic exchange of SAF-A:RNA complex" does not really provide an explanation.

    We have expanded the text in this section to explain this phenotype in greater detail (p7 lines 216-218).

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    Referee #3

    Evidence, reproducibility and clarity

    The authors investigate an interesting question focussed on whether nuclear RNA from the previous cell cycle is present in the subsequent G1. It turns out that this is more complex than expected with some classes of RNA being inherited whilst others are not. SAF-A or HNRNPU had been implicated in this process but the authors suggest that its role is limited.

    Figure 1

    In panel A the authors write on image SAF-A-mCh. What does this refer to? Panel B and other panels can the authors present this data as a boxplot or distribution plot to get a better feel of the data distribution spread. Presumably labelled RNAs are naturally turned over. Have the authors considered that some loss of signal could be because of this? Panel E, have the authors considered labelling RNA before RO3306 treatment? What effect would this have? Shouls TI be added before RO3306 washout? Also, it is unclear what the arrows are pointing at. In panel F there is a difference between the red and blue experiments. In the methods the authors say that inhibition was for either 1.5 or 2 h. Is this the source of the difference?

    Figure 2

    In panel A there are clear differences between the cell lines. Is it right to compare them? Particularly the GRID-seq vs diMARGI? B, how relevant is it focussing on the "42" overlapping RNAs? In my mind this is not very informative. D-E, at a glance it is not clear that E is an expanded view of D. It might be easier if the panels were at same height.

    Figure 3

    Is it correct to describe IAA treated degron cells as a KO? I also could not see a WB showing how complete SAF-A KD was. 2 h treatment seems quite short, is this enough time to obtain sufficient knock down? How heterogenous is SAF-A KD in the cell population? Previous studies have shown that SAF-A does not like being tagged. How certain are the authors that these cells behave typically?

    Figure 4

    I'm struggling with the heading, and wonder if this is not supported by the data. Similarly the final sentence "The highly dynamic exchange of SAF-A:RNA complex" does not really provide an explanation.

    Significance

    This is a clear well undertaken study that has made some interesting observations, which will inform future studies.

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    Referee #2

    Evidence, reproducibility and clarity

    Peer review of manuscript "Differential nuclear import determines lncRNA inheritance following mitosis" by Blower et al.

    Section A: Evidence, reproducibility, and clarity

    Summary:

    In this manuscript, Blower and colleagues examine the fate of nuclear RNAs following cell division. Using cell synchronization methods combined with RNA labeling with EU, the authors show that some of the nuclear RNAs synthesized in the previous cell cycle are relocalized to the nucleus of the new daughter cells in G1. To assess which classes of nuclear RNAs could be 'inherited' after cell division, they used bioinformatic analyses of previous studies, and found a small group of non-coding RNAs in common. To validate a few of these, the authors used RNA FISH to quantify nuclear signals of U2, MALAT1, and NEAT1 in G2 and subsequent G1 stages, and found that only U2 seems to be relocalized to the nucleus following division. The authors then tested whether RNA inheritance could be driven by SAF-A by examining the localization of U2, MALAT1, and NEAT1 in G1 when SAF-A WT or SAF-Amutant (retained in mitosis) is present. But they found that SAF-A plays a minor role in this process. Finally, they found that for U2, nuclear import is required for its relocalization to the nucleus in G1.

    Major comments:

    The authors pose an interesting question -- how does nuclear RNA segregate following mitosis. In many ways, the results presented in this manuscript are rather preliminary. Key controls and validation are missing. Because of this, it is difficult to assess the validity of the main conclusions of the study. More specifically:

    1. The main conclusion of the manuscript ("about half of nuclear RNA is inherited by G1 cells following division") is primarily dependent on the experiment described in Fig 1A-B. The authors labeled synchronized cells with EU and quantified nuclear signal after release from synchronization. However, key controls are missing. What is the synchronization efficiency of the RO3306 treatment? How many cells in their acquired fields of cells are in G2 vs in other cell cycle stages? Following their drug release, what percentage of the synchronized cells have undergone telophase? What is the potential error rate in identifying the cell cycle stage using their visual imaging analysis? Without these key controls, it is unclear how to interpret the data presented in Fig 1B.
    2. The use of transcriptional inhibitors in Fig 1 is really nice and is important for showing that it's not due to new transcription following mitosis. Well done!
    3. One potential mechanism that could explain the observed 25% relocalized nuclear RNA is through passive diffusion. That is, a proportion of molecules that are randomly diffusing during mitosis get trapped inside the newly formed nuclear membrane in early G1. This would be considered noise, and not a specific process that actively relocalizes nuclear RNA back into the nucleus. However, the authors' assay does not have a measure of the noise in their system. One potential experiment that may help quantify this noise is to express GFP in their cells, perform the experiment described in Fig 1A, and quantify the nuclear signal after telophase. This quantification would be the lower bound of the random process. A similar experiment with GFP-NLS could be performed to assess the upper bound of the 'inherited' molecules after mitosis. Without this type of control to quantify noise/random diffusion levels, it is unclear how much of the 25% EU signal that the authors detect is specific to the process they are testing.
    4. Related to the comment 1 and 2, EU labeling for 3 hrs in G2 cells would label ALL transcribed RNA, which would include mature mRNAs that will be translated in the cytoplasm. That is, this method is not specific to labeling nuclear RNAs only. How much of their signal is from mRNAs that got trapped inside the newly formed nuclear membrane? One way to test this is to measure the nuclear EU signal at later time points following telophase. Presumably, the nuclear transport mechanism would lead to export of non-nuclear RNAs and only the retained nuclear RNAs would contribute to the signal.
    5. To identify nuclear RNAs that could be relocalized following mitosis, the authors analyzed data from "two different studies using different methodologies and a total of three different cell lines". From this analysis, the authors "found very little overlap in the chromatin-bound RNAs identified in these studies (Fig 2A)". This analysis seems fraught with problems. What is the rationale for using these studies? How valid is it to compare results from different methodologies and from different cell lines from the DLD-1 cells used in this study? A known problem of assessing chromatin-bound RNAs is that the level of contamination from cytoplasmic RNAs is highly variable and highly dependent on the assay. Indeed some of the most common contaminants of nuclear RNA assays are sn-, and sno-RNAs, and these are the main classes of RNA that the authors identified as common among the three data sets. What validation was used to assess whether these are the common noise/contaminants in the data? One experimental validation that can be performed is biochemical fractionation of EU labeled cells, which would allow for fractionating nuclear from cytoplasmic RNA. The same problems arise with the analysis shown in Fig 3C when comparing SAF-A RIP-seq with this merged list of chromatin bound RNAs.
    6. Throughout the manuscript, the authors pose their findings as "RNA inheritance" following mitosis. However, this terminology is misleading. In fact, unless RNAs are lost/kicked out of the cell as they divide, aren't all RNAs inherited following cell division since they are present in the new daughter cells? Instead, what the authors mean is that some nuclear RNAs retain their function following cell division by relocalizing back into the nucleus in the new G1 cells, whereas other nuclear RNAs are unable to relocalize into the nucleus, and then presumably turned over by degradation process. The authors should take better care of their terminology throughout the manuscript.

    Minor comments:

    1. In all of the figures showing quantification of nuclear EU/FISH signal, the colors (red v blue) are not described (not found in the legend or methods). Presumably they are biological replicates, but this should be clearly stated.
    2. Is figure 2E the same data presented in Fig 2D but in different y-axis? If so, state clearly
    3. Figure 3A. This experiment is using the SAF-A-AID-mCherry system. Therefore the label in Fig 3A should be SAF-A-KD (Knockdown) instead of KO (knockout)
    4. Typo in Fig 4B y-axis. It should be "Chromatin-localized SAF-A" instead of "Chromain-localized SAF-A"
    5. The methods section indicate the "precise N or replicates in indicated figure legends" but none of the figure legends have the N values listed.

    Significance

    General assessment:

    The authors pose an interesting question -- how does nuclear RNA segregate following mitosis. However, the results presented in this manuscript are rather preliminary. Key controls and validation are missing. Because of this, it is difficult to assess the validity of the main conclusions of the study.

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    Referee #1

    Evidence, reproducibility and clarity

    This study follows up on the authors' recent work showing entry into mitosis is associated with RNA removal from condensing chromosomes and investigates whether the same RNAs that were removed are reincorporated into G1 chromosomes. The study is timely and simple yet elegant. The authors use pulse chase experiments to follow the distribution of bulk RNAs in synchronized then released cells, showing a subset of labeled RNAs are reincorporated into chromatin. This process is selective, as the authors demonstrate some RNAs are more likely to reassociate with G1 chromosomes than others. The authors go on to use various small molecule inhibitors to provide mechanistic insights showing transcription is not required for RNA inheritance, but nuclear pore components (importin-B) are.

    The study is well conceived and executed. I recommend the following relatively minor revisions.

    Major point:

    1. The authors rely upon the redistribution of RNA to measure the inheritance of extant RNAs following cell cycle release. Blocking transcription nicely shows new synthesis is not required for this inheritance. This is also consistent with the idea any newly synthesized RNA would be 'dark,' or not EU labeled, but the transcription inhibitor experiments are critical controls and nicely done. As hinted at the end of their discussion, however, a lack of RNA localizing to G1 chromosomes could be formally attributable to differential RNA stability. Might altered RNA stability of NEAT1, MALAT1, or U2 also contribute to the observed altered localizations upon interphase reentry? The authors could use qPCR or measure RNA half-life to test this possibility. These data would nicely compliment the authors' existing FISH experiments and allow them to specifically argue for differential RNA localization.

    Minor Points:

    1. The authors examine published datasets identifying RNA associated with chromatin and state the reason why these data show little overlap is "primarily attributable to purification methodology." This statement seems speculative, and its basis seems unclear.
    2. The SAF-A-AA experiments failed to reveal insight into mechanisms of RNA sorting, although they do suggest the AA construct functions as a gain-of-function due to a) increased RNA reincorporated into chromosomes b) dramatic increase of chromosome targeting of SAF-A. These effects make it difficult to interpret the SAF-A-AA data. Related to this point, the analysis of altered RNA distributions relative to SAF-A is underdeveloped. Because the authors only examined one lncRNA (MALAT1), the conclusion that "forced retention of SAF-A on mitotic chromatin does not lead to an increase in the nuclear inheritance of specific transcripts" seems like an overstatement.
    3. The authors find the U2 spliceosomal RNA is preferentially inherited. Might they speculate why this would be advantageous?
    4. Optional: it would be exciting to test the significance of U2 RNA inheritance
    5. For Figure 1, please add statistics to figures and legend; add N=cells examined.
    6. For Figure 2, single channel panel of U2 RNA should be added. Figure 2E seems to reproduce the same data shown in Figure 2D (right-most columns) shown with different axes.
    7. Figure 3, it is unclear why the authors selected MALAT1 for analysis, but not NEAT1 (or the single (unlabeled) antisense RNA also enriched in the SAF-A IP (figure 2C).
    8. Figure 4B, please add statistics to figure and legend.
    9. Methods: in their description of the published lists of chromatin-bound RNAs, the authors should cite those works and provide a data availability statement with the associated GEO

    Significance

    General assessment: (strengths) The study is timely and simple yet elegant. It is well conceived and executed. In general, the conclusions are supported by the data. The study provides insight into a fundamental basic science process likely of interest to a broad readership. (weaknesses) The authors do provide some mechanistic insight into how RNAs are inherited, although this mechanism will need to be further developed in future studies. The role of SAF-A is unclear. Because those experiments did not produce a clear effect, they seem distracting. The idea that specific RNAs are sorted is exciting, but as yet we do not know how this sorting happens. Future work examining the importance of RNA inheritance should be prioritized. Measuring RNA abundance of the different analyzed RNAs (NEAT1, MALAT1, vs U2) should be added to the current manuscript.

    Advance: This study follows up on the authors' recent work showing entry into mitosis is associated with RNA removal from condensing chromosomes and investigates whether the same RNAs that were removed are reincorporated into G1 chromosomes.

    Audience: The study provides insight into a fundamental basic science process likely of interest to a broad readership.

    Describe your expertise: basic cell biology, RNA localization

  6. Version published to 10.1101/2022.10.06.511141 on bioRxiv