N -6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5′ Splice Site Motifs

  1. Eliza S. Lee
  2. Harrison. W. Smith
  3. Yifan E. Wang
  4. Sean S. J. Ihn
  5. Leticia Scalize de Olivera
  6. Nevraj S. Kejiou
  7. Yijing L. Liang
  8. Syed Nabeel-Shah
  9. Robert Y. Jomphe
  10. Shuye Pu
  11. Jack F. Greenblatt
  12. Alexander F. Palazzo

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Abstract

In humans, misprocessed mRNAs containing intact 5′ Splice Site (5′SS) motifs are nuclear retained and targeted for decay by ZFC3H1, a component of the Poly(A) Exosome Targeting complex, and U1-70K, a component of the U1 snRNP. In S. pombe , the ZFC3H1 homolog, Red1, binds to the YTH-domain containing protein Mmi1 to target certain RNA transcripts to nuclear foci for nuclear retention and decay. Here we show that YTHDC1 and YTHDC2, two YTH domain-containing proteins that bind to N-6-methyladenosine (m6A) modified RNAs, interact with ZFC3H1 and U1-70K, and are required for the nuclear retention of mRNAs with intact 5′SS motifs. Disruption of m6A deposition inhibits both the nuclear retention of these transcripts and their accumulation in YTHDC1-enriched foci that are adjacent to nuclear speckles. Endogenous RNAs with intact 5′SS motifs, such as intronic polyadenylated transcripts, tend to be m6A-modified at low levels. Thus, m6A modification acts in a conserved quality control mechanism that targets misprocessed mRNAs for nuclear retention and decay.

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  1. Version published to 10.1101/2023.06.20.545713v3 on bioRxiv
  2. Version published to 10.1101/2023.06.20.545713v2 on bioRxiv
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    Reply to the reviewers

    We thank the reviewers for their thoughtful comments. Here we provide a point-by-point response to their reviews. All additional experiments that are present in the revised manuscript, or that we plan to include in the final manuscript, are numbered.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The concept introduced by this paper is exciting and novel. However, the current paucity of presented data can lead to incorrect interpretations of the findings and speculations that might not hold true after a more rigorous assessment of the observed phenomenon.

    The premise of this study builds upon an interaction between the PAXT complex and nuclear YTH domain containing proteins. However, figures 1B and C should be improved. The interacting band for the ZFC3H1 presented in panel B does not seem to match the size of the construct used in panel C. Is the Flag version of ZFC3H1 expressing a smaller isoform for this protein? __

    The reviewer is correct in that endogenous ZFC3H1 (which migrates at 250kD with a minor band at 150kD, see Figure 1B in the initial manuscript) appears to differ from the FLAG-tagged construct as expressed from a plasmid transfected in HEK293 cells (which migrates as two bands at 180kD and 200kD, see Figure 1C in the initial manuscript). For the endogenous protein, the predicted molecular weight is 226kD and the 250kD band disappears when cells are transduced with lentivirus containing shRNAs against ZFC3H1 (see Figure 4A in the initial manuscript), indicating that it is the correct protein. Both the 250kD endogenous protein (*) and the 200kD overexpressed protein (**) in transfected HEK293 and U2OS cells are detected in immunoblots using anti-ZFC3H1 antibodies (see Figure 1 in this document) indicating that the over-expressed protein is indeed ZFC3H1.

    [ Figure 1]

    ____Figure 1. Molecular Weight Size Comparison of Endogenous ZFC3H1 and FLAG-ZFC3H1 (1-1233). ____Lysates from HEK293 and U2OS that were either untransfected or transfected with FLAG-ZFC3H1 (1-1233) plasmid. We labelled the bands corresponding to the endogenous ZFC3H1 “*” and FLAG-ZFC3H1 (1-1233) “**”.

    We have sequenced the plasmid, and discovered that it contains an additional sequence inserted within the middle of the ZFC3H1 cDNA with a premature stop codon. As such, the version of the protein that is expressed from the plasmid only contains amino acids 1-1233 of the endogenous protein and is missing amino acids 1234-1989. The deleted region only contains TPR repeats, and is not known to interact with any of the well characterized interactors of ZFC3H1 (Wang, Nuc Acid Res 2021, Figure 3). We have renamed this construct FLAG-ZFC3H1 (1-1233). Given these new considerations, our results are consistent with the idea that the N-terminal portion of ZFC3H1 interacts with U1-70K, YTHDC1 and YTHDC2. We will change the text to reflect this.

    We are currently in the process of deleting the small insertion to obtain a plasmid that encodes a full length version of human ZFC3H1. For the final manuscript:

    Experiment #1) We will repeat the co-immunoprecipitation experiment with the full length FLAG-ZFC3H1 to determine whether it interacts with YTHDC1 and YTHDC2. This will take a few weeks.

    __Also, the YTHDC1-2 interaction in panel C is not as convincing considering the negative controls lane show some degree of binding. __

    Although the reviewer is correct that there is substantial background binding in the YTHDC1 immunoblot, we disagree with their characterization of the results with the YTHDC2 immunoblot (see Figure 1B-C in the initial manuscript). In the new manuscript we have included:

    Experiment #2) A new co-immunoprecipitate of the FLAG-tagged ZFC3H1 (1-1233) from HEK293 cells under more stringent conditions where the background level of YTHDC1 binding to beads is negligible. We have already completed this experiment (see Figure 1D in the revised manuscript).

    __Additionally, can the authors test if their RNaseA treatment worked? __

    In the new manuscript we have included:

    Experiment #3) A new co-immunoprecipitate of FLAG-YTHDC1 immunoblotted for eIF4AIII from HEK293 cell lysates. We find that without RNAse, there is some eIF4AIII in the precipitates but that the levels diminish substantially after RNAse A/T1 treatment. We have already completed this experiment (see Figure 1B in the preliminary revised manuscript).

    __Why do you need 18 hours to observe the nuclear export of your modifiable construct when inhibiting METTL3 in figure 3? Is it possible that your observation is secondary to phenotypes these cells develop as a result of blocking METTL3? __

    We treated cells for this period of time so that during the expression of the reporter, all of the newly synthesized mRNA is expressed in the absence of m6A methyl transferase activity. For shorter treatment times, it is unclear whether the bulk of the reporter mRNA, which would be synthesized before the treatment, would lose any pre-existing m6A marks, making a negative result hard to interpret. Previously we found that although 50% of intronic polyadenylated (IPA) transcripts from our reporters are rapidly degraded, about 50% are stable and are nuclear retained over extended periods of time (see Lee at al., PLOS ONE 2015; https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122743 Figure 3B-G). We believe that the bulk of the reporter mRNA that we are visualizing is stable and accumulates in the nucleus. Given that METTL3-depletion inhibits nuclear retention and that versions of the IPA reporter that lack m6A modification motifs are exported, we think that the most likely interpretation of the 18 hour STM2457 treatment experiments is that the lack of methyltransferase activity had a direct effect, rather than an indirect effect, on nuclear retention. We would be open to performing more experiments if the editors insist, however we ordered STM2457 four weeks ago and it has yet to arrive from Sigma-Millipore. Performing this experiment may substantially delay our ability to resubmit the manuscript in a timely manner.

    __Is ALKBH5 nuclear and/or cytoplasmic in the cell system used? __

    According to The Human Protein Atlas, ALKBH5 is predominantly nuclear in U2OS cells, with some present in the cytoplasm (https://www.proteinatlas.org/ENSG00000091542-ALKBH5/subcellular#human).

    In the revised manuscript we have included:

    Experiment #4) Data from subcellular fractionation demonstrating that ALKBH5 is present in both the nucleus and cytoplasm that we have already performed (see Figure 4J in the preliminary revised manuscript).

    __Reviewer #1 (Significance (Required)):

    The study is highly significant __

    ------

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary: In the manuscript by Lee et al. entitled "N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5'Splice Site Motifs", the authors provide evidence that m6A modifications within specific regions of transcripts can confer nuclear retention. These results are important because they add to our understanding of how m6A modifications can contribute to post-transcriptional regulation. Although the authors do not quite come out and say this, data seem to be accumulating to suggest that the location of the m6A modifications within a given transcript can dictate the functional consequences of those modifications.__

    We thank the reviewer for pointing this out. We have included a few sentences in the new preliminary revised manuscript pointing out that the location of the m6A modification in IPA transcripts, with respect to intact 5’SS and poly(A) signals, may play a role in promoting nuclear retention.

    __The current work builds on previous findings from these authors identifying factors critical for retention of intronic polyadenylated (IPA) transcripts. The present study identified m6A modification as one of the signals for the retention of such transcripts. The authors use reporters for their analysis and also examine validated endogenous IPA transcripts. The data presented supports the conclusions albeit they show a surprising finding for one of the m6A erasers, ALKBH5. However, there is some controversy over the mechanism by which ALKBH5 functions and whether the m6A mark is truly reversible, so these results may continue to add to this point of view.

    Major Comments: One experiment that might add to the argument would be overexpression of Mettl3 as compared to catalytically inactive Mettl3. The prediction would be that the reporter transcript with intact DRACH sequences would be even more retained in the nucleus in a manner that depends on Mettl3 catalytic activity. For some of the data presented, the reporter is already wholly nuclear so no difference could be detected, but in the U2OS cells shown in Figure 2B, it appears that an increase in nuclear localization might be evident. Such an experiment would add an orthogonal approach to demonstrate that the methylation by Mettl3 is required for retention. If such an experiment would work with the endogenous IPA transcripts shown in Figure 4, but these transcripts may already be too nuclear to detect any increase in nuclear retention.

    __

    We have performed two experiments that try to address this but they gave negative results:

    Experiment #5) We have over-expressed wildtype and a methyl transferase mutant FLAG-METTL3 and assessed the nuclear export/retention of ftz-Δi-5’SS mRNA. There was no effect (see Figure 2 in this document).

    [Figure 2]

    __Figure 2. Over-expression of METTL3 does not increase the nuclear retention of ftz-Δi-5’SS. __U2OS cells were co-transfected with ftz-Δi-5’SS reporter and either FLAG-METTL3 or FLAG-METTL3-D395A, which lacks methyl-transferase activity (Wang, Mol Cell 2016). Cells were fixed, stained for ftz mRNA by fluorescent in situ hybridization and METTL3 using anti-FLAG antibodies. The nuclear and cytoplasmic distribution of ftz mRNA was quantified as described in the manuscript. Note that this is the average of one independent experiment (each bar consisting of the average of at least 50 cells). We plan to repeat this two more times, but we anticipate that these will show the same result.

    We could include this negative data as a supplemental figure. We believe that there are two possible reasons for this experimental result. First, as the reviewer points out, the reporter transcripts are already too nuclear to detect any significant change. Second, METTL3 is part of a larger complex that includes several proteins including METTL14, WTAP and potentially other proteins (for example see Covelo-Molares, Nuc Acid Res 2021). We may need to co-express all of these proteins to see an effect.

    Experiment #6) We have also expressed versions of ftz-Δi and ftz-Δi-5’SS mRNA with optimized m6A modification (i.e. DRACH) motifs (AGACT) to enhance methylation (“e-m6A-ftz”). We only observed a slight increase in nuclear retention but it is not significant (see Figure 2A,C in the revised manuscript).

    Again, this result could be explained by the fact that the reporter is too nuclear to detect any significant increase in retention. We had originally performed this in parallel with the no-m6A-ftz-Δi-5’SS reporters but did not report this negative data in the original manuscript.

    __Some rather minor changes to the presentation of the data could enhance the impact of this study.

    Specific Comments:

    The primary question in this manuscript is comparing reporters with m6A site (intact DRACH sequences) to those without. For this reason, organizing the data to the +/- DRACH sites are adjacent to one another might make the most sense. This point is evident in Figure 1C where perhaps simply changing the order of the bars presented to put the ones directly compared adjacent would be preferable. Then the p-value would compare sets of data directly adjacent to one another. __

    We thank the reviewer for this suggestion and we have made these changes to the figures in the preliminary revised manuscript.

    __While the authors show representative fields/cells for most assays, they do an excellent job of providing quantitation as well. One exception is Figure 3D, which shows a single cell image for the most key panel (the 5'SS-containing reporter upon Mettl3 depletion). If there is not a field with more cells, the authors could create a montage. __

    In the revised manuscript, we have replaced this image with one containing multiple cells expressing the reporter.

    __Minor Comments:

    Figure presentation:

    The text in a number of the figures is VERY small (Figures 1B,1C, and 4A) for example. __

    We have fixed this in the new manuscript.

    __Figure 3A includes the label "shRNA:" at the top, but these cells are treated with Mettl3 inhibitor and there does not appear to be any shRNA employed, so this seems like a labeling error. __

    We have fixed this in the new manuscript.

    __In Figure 3C, the immunoblot of Mettl3, there are three bands that all disappear completely upon knockdown of Mettl3- are these all Mettl3? This should at least be mentioned in the legend and perhaps indicated in the figure. The authors do mention in the text employing shRNAs to target multiple Mettl3 isoforms, so likely this is the case. __

    We have clarified these issues in the new manuscript.

    __Minor points (some really minor to just polish the presentation for clarity):

    The word "since" should only be used if there is a time element- otherwise the word "as" is preferable.

    For example on p. 4, the sentence: "Since inhibition of mRNA export typically enhances the nuclear retention of RNAs with intact 5'SS motifs (Lee et al. 2020),.." would more precisely read "As inhibition of mRNA export typically enhances the nuclear retention of RNAs with intact 5'SS motifs (Lee et al. 2020),..". __

    We thank the reviewer for pointing this out. We have fixed this issue in the revised manuscript.

    __Reviewer #2 (Significance (Required)):

    Summary: In the manuscript by Lee et al. entitled "N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5'Splice Site Motifs", the authors provide evidence that m6A modifications within specific regions of transcripts can confer nuclear retention. These results are important because they add to our understanding of how m6A modifications can contribute to post-transcriptional regulation. Although the authors do not quite come out and say this, data seem to be accumulating to suggest that the location of the m6A modifications within a given transcript can dictate the functional consequences of those modifications.

    This study would be of significant interest to those that study gene expression in any context as well as cell biologists as the data add to our understanding of export of mRNA from the nucleus. This work also adds to our understanding of the biological consequences of m6A modification, which is an area of significant interest. In my opinion, the authors could make a broader conclusion that we do, which is that the location of the modification significantly dictates function- an extension of previous findings mostly focused on processed mRNA transcripts. __

    -------

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Quality control of mRNA is vital for all types of cells. In eukaryotic cells, nuclear export of misprocessed mRNAs containing the 5' splice site is prevented. In this manuscript, Lee and colleagues demonstrate that the nuclear retention of intronic polyadenylated transcripts is dependent on m6A modification. Based on the results shown in yeast, they perform immunoprecipitation experiments and demonstrate the interaction between ZFC3H1, a component of the PAXT complex, and YTHDC1 and YTHDC 2, nuclear YTH RNA-binding proteins that recognize m6A-modified transcripts. The study also shows the interaction of U1-70K with YTHDC1 and with ZFC3H1. Depletion of YTHDC1/2 prevents the nuclear retention of IPA transcripts. Additionally, CLIP-seq analysis is performed, demonstrating that m6A modification is enriched around the 5' splice site motif and the 3' polyadenylation site in IPAs. From these observations, they conclude that m6A modification contributes to the quality control of mRNA by promoting nuclear retention of misprocessed transcripts.

    Major Points

    1. The interaction between ZFC3H1 and YTHDC1 is clearly shown by immunoprecipitation of FLAG-tagged YTHDC1 in Figure 1B. However, the co-purification of YTHDC1 with FLAG-tagged ZFC3H1 in Figure 1C is rather ambiguous. Additionally, the immunoprecipitated samples do not appear to show signals corresponding to FLAG-tagged ZFC3H1, making it unclear if the immunoprecipitation is working. It is essential to provide a better quality result to clarify these observations. __

    Please see our responses to reviewer #1. We have repeated the co-immunoprecipitation of FLAG-ZFC3H1 (1-1233) with YTHDC1 under more stringent conditions and have reduced the background binding (see Figure 1B and D in the new manuscript). We have also determined why the FLAG-ZFC3H1 is smaller than expected as the construct contains a premature stop codon. As explained above, we are in the midst of generating a full-length FLAG-ZFC3H1 and we plan to repeat the co-immunoprecipitation with this new construct.

    2. While the authors demonstrate that the m6A modification is dispensable for the targeting of IPA reporter transcripts to the nuclear speckles, it would be valuable to investigate whether m6A is required for their exit from the nuclear speckles. Do reporter transcripts with m6A motifs remain in the nuclear speckles at later time points?

    We have now analyzed the colocalization of nuclear speckles (SC35) with ftz-Δi-5’SS, which contains both a 5’SS and DRACH motifs, and no-m6A-ftz-Δi-5’SS, which contains a 5’SS but lacks DRACH motifs, at steady state – i.e. after 18-24 hours of transfection (as opposed to at early time points as shown in Figure 2D-E of the initial manuscript). Unexpectedly, we see that both mRNAs continue to colocalize with nuclear speckles, although the no-m6A-ftz-Δi-5’SS mRNA is well exported from the nucleus and its signal in nuclear speckles is faint (see Figure 2F-H in the new manuscript).

    Previously, we observed that *ftz-Δi-5’SS *required the 5’SS motif to remain in nuclear speckles at these later time points (Lee *PLOS ONE *2015 and Lee RNA 2022). Upon closer inspection, *ftz-Δi-5’SS *mRNA also accumulates in additional nuclear foci that are not SC35-positive. Our new results may indicate that m6A marks promote the transfer of mRNAs from nuclear speckles to other foci, but more data is required to make a firm statement. Given this, we plan to conduct further experiments which may take a month to complete:

    Experiment #7) We are now assessing whether these additional *ftz-Δi-5’SS *foci correspond to either YTHDC-positive foci which were previously shown to partially overlap nuclear speckles and sequester m6A-rich mRNAs (Cheng Cancer Cell 2022), or “pA+ RNA foci” which accumulate MTR4/ZFC3H1-targetted RNAs when the nuclear exosome is inhibited (Silla Cell Reports 2018). These foci are enriched in ZFC3H1. We plan on co-staining ftz-Δi, ftz-Δi-5’SS, no-m6A-ftz-Δi and no-m6A-ftz-Δi-5’SS with SC35, YTHDC1 and ZFC3H1 to determine whether m6A may help to transfer mRNAs from nuclear speckles to YTHDC1 or ZFC3H1-enriched foci.

    __3. Figures 5B and 5C suggest that ZFC3H1 is required for the degradation of IPA transcripts. However, the range of the vertical axis is inappropriate and it is difficult to assess the extent of the increase in expression levels. Please adjust the vertical axis range for improved clarity. __

    We thank the reviewer for the feedback we have added additional graphs with an expanded vertical axis to demonstrate that ZFC3H1 is required for the degradation IPA transcripts.

    __Minor Points

    1. page 4, line 2 "RNAse" should be corrected to "RNase".__

    We thank the reviewer for catching this error. We have fixed this.

    __ 2. page 7, line 5: Is the statement "prevents the nuclear export and decay of non-functional and misprocessed RNAs" correct? m6A modification promotes the decay of such RNAs. __

    We thank the reviewer for pointing this out. We have altered the text to clarify that m6A promotes decay.

    __3. Figure 2E: ftz-∆i should be ftz-∆i-5'SS. __

    We thank the reviewer for catching this error. We have fixed this.

    __4. Figure 5A: It would be helpful to indicate the number of IPA transcripts analyzed. __

    We have included this information.

    __Reviewer #3 (Significance (Required)):

    Overall, the work is sound and generally well-controlled. This study advances our understanding of the quality control of misprocessed transcripts in higher eukaryotes. This reviewer suggests a few points for clarification or improvement. __

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    Referee #3

    Evidence, reproducibility and clarity

    Quality control of mRNA is vital for all types of cells. In eukaryotic cells, nuclear export of misprocessed mRNAs containing the 5' splice site is prevented. In this manuscript, Lee and colleagues demonstrate that the nuclear retention of intronic polyadenylated transcripts is dependent on m6A modification. Based on the results shown in yeast, they perform immunoprecipitation experiments and demonstrate the interaction between ZFC3H1, a component of the PAXT complex, and YTHDC1 and YTHDC 2, nuclear YTH RNA-binding proteins that recognize m6A-modified transcripts. The study also shows the interaction of U1-70K with YTHDC1 and with ZFC3H1. Depletion of YTHDC1/2 prevents the nuclear retention of IPA transcripts. Additionally, CLIP-seq analysis is performed, demonstrating that m6A modification is enriched around the 5' splice site motif and the 3' polyadenylation site in IPAs. From these observations, they conclude that m6A modification contributes to the quality control of mRNA by promoting nuclear retention of misprocessed transcripts.

    Major Points

    1. The interaction between ZFC3H1 and YTHDC1 is clearly shown by immunoprecipitation of FLAG-tagged YTHDC1 in Figure 1B. However, the co-purification of YTHDC1 with FLAG-tagged ZFC3H1 in Figure 1C is rather ambiguous. Additionally, the immunoprecipitated samples do not appear to show signals corresponding to FLAG-tagged ZFC3H1, making it unclear if the immunoprecipitation is working. It is essential to provide a better quality result to clarify these observations.
    2. While the authors demonstrate that the m6A modification is dispensable for the targeting of IPA reporter transcripts to the nuclear speckles, it would be valuable to investigate whether m6A is required for their exit from the nuclear speckles. Do reporter transcripts with m6A motifs remain in the nuclear speckles at later time points?
    3. Figures 5B and 5C suggest that ZFC3H1 is required for the degradation of IPA transcripts. However, the range of the vertical axis is inappropriate and it is difficult to assess the extent of the increase in expression levels. Please adjust the vertical axis range for improved clarity.

    Minor Points

    1. page 4, line 2 "RNAse" should be corrected to "RNase".
    2. page 7, line 5: Is the statement "prevents the nuclear export and decay of non-functional and misprocessed RNAs" correct? m6A modification promotes the decay of such RNAs.
    3. Figure 2E: ftz-∆i should be ftz-∆i-5'SS.
    4. Figure 5A: It would be helpful to indicate the number of IPA transcripts analyzed.

    Significance

    Overall, the work is sound and generally well-controlled. This study advances our understanding of the quality control of misprocessed transcripts in higher eukaryotes. This reviewer suggests a few points for clarification or improvement.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: In the manuscript by Lee et al. entitled "N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5'Splice Site Motifs", the authors provide evidence that m6A modifications within specific regions of transcripts can confer nuclear retention. These results are important because they add to our understanding of how m6A modifications can contribute to post-transcriptional regulation. Although the authors do not quite come out and say this, data seem to be accumulating to suggest that the location of the m6A modifications within a given transcript can dictate the functional consequences of those modifications.

    The current work builds on previous findings from these authors identifying factors critical for retention of intronic polyadenylated (IPA) transcripts. The present study identified m6A modification as one of the signals for the retention of such transcripts. The authors use reporters for their analysis and also examine validated endogenous IPA transcripts. The data presented supports the conclusions albeit they show a surprising finding for one of the m6A erasers, ALKBH5. However, there is some controversy over the mechanism by which ALKBH5 functions and whether the m6A mark is truly reversible, so these results may continue to add to this point of view.

    Major Comments: One experiment that might add to the argument would be overexpression of Mettl3 as compared to catalytically inactive Mettl3. The prediction would be that the reporter transcript with intact DRACH sequences would be even more retained in the nucleus in a manner that depends on Mettl3 catalytic activity. For some of the data presented, the reporter is already wholly nuclear so no difference could be detected, but in the U2OS cells shown in Figure 2B, it appears that an increase in nuclear localization might be evident. Such an experiment would add an orthogonal approach to demonstrate that the methylation by Mettl3 is required for retention. If such an experiment would work with the endogenous IPA transcripts shown in Figure 4, but these transcripts may already be too nuclear to detect any increase in nuclear retention.

    Some rather minor changes to the presentation of the data could enhance the impact of this study.

    Specific Comments:

    The primary question in this manuscript is comparing reporters with m6A site (intact DRACH sequences) to those without. For this reason, organizing the data to the +/- DRACH sites are adjacent to one another might make the most sense. This point is evident in Figure 1C where perhaps simply changing the order of the bars presented to put the ones directly compared adjacent would be preferable. Then the p-value would compare sets of data directly adjacent to one another.

    While the authors show representative fields/cells for most assays, they do an excellent job of providing quantitation as well. One exception is Figure 3D, which shows a single cell image for the most key panel (the 5'SS-containing reporter upon Mettl3 depletion). If there is not a field with more cells, the authors could create a montage.

    Minor Comments:

    Figure presentation:

    The text in a number of the figures is VERY small (Figures 1B,1C, and 4A) for example.

    Figure 3A includes the label "shRNA:" at the top, but these cells are treated with Mettl3 inhibitor and there does not appear to be any shRNA employed, so this seems like a labeling error.

    In Figure 3C, the immunoblot of Mettl3, there are three bands that all disappear completely upon knockdown of Mettl3- are these all Mettl3? This should at least be mentioned in the legend and perhaps indicated in the figure. The authors do mention in the text employing shRNAs to target multiple Mettl3 isoforms, so likely this is the case.

    Minor points (some really minor to just polish the presentation for clarity):

    The word "since" should only be used if there is a time element- otherwise the word "as" is preferable.

    For example on p. 4, the sentence: "Since inhibition of mRNA export typically enhances the nuclear retention of RNAs with intact 5'SS motifs (Lee et al. 2020),.." would more precisely read "As inhibition of mRNA export typically enhances the nuclear retention of RNAs with intact 5'SS motifs (Lee et al. 2020),..".

    Significance

    Summary: In the manuscript by Lee et al. entitled "N-6-methyladenosine (m6A) Promotes the Nuclear Retention of mRNAs with Intact 5'Splice Site Motifs", the authors provide evidence that m6A modifications within specific regions of transcripts can confer nuclear retention. These results are important because they add to our understanding of how m6A modifications can contribute to post-transcriptional regulation. Although the authors do not quite come out and say this, data seem to be accumulating to suggest that the location of the m6A modifications within a given transcript can dictate the functional consequences of those modifications.

    This study would be of significant interest to those that study gene expression in any context as well as cell biologists as the data add to our understanding of export of mRNA from the nucleus. This work also adds to our understanding of the biological consequences of m6A modification, which is an area of significant interest. In my opinion, the authors could make a broader conclusion that we do, which is that the location of the modification significantly dictates function- an extension of previous findings mostly focused on processed mRNA transcripts.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The concept introduced by this paper is exciting and novel. However, the current paucity of presented data can lead to incorrect interpretations of the findings and speculations that might not hold true after a more rigorous assessment of the observed phenomenon.

    The premise of this study builds upon an interaction between the PAXT complex and nuclear YTH domain containing proteins. However, figures 1B and C should be improved. The interacting band for the ZFC3H1 presented in panel B does not seem to match the size of the construct used in panel C. Is the Flag version of ZFC3H1 expressing a smaller isoform for this protein? Also, the YTHDC1-2 interaction in panel C is not as convincing considering the negative controls lane show some degree of binding. Additionally, can the authors test if their RNaseA treatment worked?

    Why do you need 18 hours to observe the nuclear export of your modifiable construct when inhibiting METTL3 in figure 3? Is it possible that your observation is secondary to phenotypes these cells develop as a result of blocking METTL3?

    Is ALKBH5 nuclear and/or cytoplasmic in the cell system used?

    Significance

    The study is highly significant

  7. Version published to 10.1101/2023.06.20.545713v1 on bioRxiv