Aurora A mediated new phosphorylation of RAD51 is observed in Nuclear Speckles

  1. Mohamad Alaouid
  2. Parfait Kenfack Ymbe
  3. Vanessa Philippot-Ménil
  4. Gwennina Cueff
  5. Alexandre Demeyer
  6. Damien Marquis
  7. Nizar Ayadi
  8. Fabrice Fleury
  9. Houda Benhelli-Mokrani

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Abstract

To maintain its genome integrity, the cell uses complementary and orchestrated processes, among which is the DNA Damage Response. Pre mRNA maturation is an essential step of the DNA damage response that provides an adapted proteome in order to face genotoxic stress. We describe here a new phosphorylation of the RAD51 recombinase, on its Ser97 residue. This new Aurora A mediated RAD51 phosphorylation modulates its in vitro activity evaluated by D-loop and polymerization assays. Using recombinant proteins, we show that RAD51 is an RNA binding protein and that the Ser97 phosphorylation modulates its RNA binding affinity in vitro . Using a specifically generated antibody we revealed that this Ser97 phosphorylation is correlated with RAD51 localization into the RNA maturation membrane less organelles, Nuclear Speckles. We describe here for the first time the presence of RAD51 DNA repair factor within the Nuclear Speckles, raising the hypothesis of a possible role for RAD51 in splicing modulation. This point is of particular interest in the context of splicing profiles modulations associated with radio and/or chemoresistance.

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    Referee #3

    Evidence, reproducibility and clarity

    In the manuscript entitled "Aurora A mediated new phosphorylation of RAD51 is observed in Nuclear Speckles", the authors unveil the Serine S97 as a novel phosphorylation site of the RAD51 recombinase and that this phosphorylation is mediated by the Aurora A kinase using a set of in vitro and in cellulo experiments. The authors also describe this phosphorylation being in the nucleus specifically in nuclear speckles where mRNA maturation and splicing occurs suggesting a role of RAD51 in the latter. The confocal microscopy images provided to test this hypothesis are convincing. However, using confocal images as well, the authors claim that RAD51 phosphorylated at S97 foci do not colocalize with the DNA damage marker -H2AX, hence a function not related to DNA damage, however the data provided does not fully support this statement. In this study, Alaouid et al, utilize mutants of RAD51 that alter S97 phosphorylation to further study its function and provide data that support RAD51 as an RNA binding protein. Overall, the manuscript shows some interesting observations that are worth pursuing however the in vitro and in cellulo results are not aligned, lack some controls, and many points should be reconsidered.

    Major comments:

    • Are the key conclusions convincing?

    Not as stated.

    Fig. 1A. The authors conclude that pS97-RAD51 favors RAD51 strand invasion capacity using the D-loop assay. Indeed, the S97D phosphomimic increased the D-loop activity 2.5-fold compared to WT RAD51. However, the S97A mutant, which is the non-phosphorylated form also increased the D-loop activity by 2-fold compared to WT (figure 1C). So, the phosphorylation or the absence of it seem to promote strand invasion. So, what is the role of the phosphorylation? There is no discussion about this. Besides, no representative image of the D-loop assay is shown, this is very important as these experiments need to be run with the relevant controls to be meaningful.

    Fig. 1D. The polymerization rate of RAD51 is probably irrelevant for its function in the absence of DNA. What do they want to get at with this assay?

    In figure 2B, the authors conclude that RAD51 phosphorylation at S97 is dynamically regulated throughout the cell cycle. Indeed, the pS97-RAD51 is well observed in asynchronous cells, and the double thymidine block time course experiment followed by PI staining shows the oscillation of the pS97-RAD51 from G1 to G2/M stage. The authors quantified the ratio of pS97-RAD51/total RAD51 to conclude this. However, it would be more accurate to also divide the above over the intensity of the loading control (tubulin) because in figure 3A for example, they quantified the ratio of pS97-RAD51/tubulin but did not consider the levels of RAD51 in their quantifications.

    In figure 3B, the authors state that pS97-RAD51 is decreased after CPT treatment and that the pS97-RAD51 foci do not localize with the DNA damage marker -H2AX. The signal of gH2AX is already weird as it does not change from Ctrl to CPT conditions (especially in HCC1806 cells). A pre-extraction of soluble protein with CSK should be used to then look at the co-localization, with the pan-staining of the two signals is difficult to draw any conclusions of colocalization. Nevertheless, the signal of RAD51 seems equal in all conditions in the images shown and it does not seem to be reduced after CPT.

    In figure 4A, the authors show that Aurora A is responsible for the S97-RAD51 phosphorylation in cellulo. Indeed, the use of an Aurora A inhibitor reduces the pS97-RAD51 signal, however, this is only true in one cell line (HCC1806) but no effect was observed in HeLa cells. Is this effect cell-specific?

    The authors find that RAD51 binds both DNA and RNA and measure the affinities of the RAD51 bearing the S97D and S97A mutations. S97D shows the highest affinity for ssDNA and RNA in Fig. 7A, B, however the opposite is true for dsDNA in Fig 7C, D. All three forms of RAD51 bind RNA although with different affinities however no error bars are shown. The description of the results does not seem accurate. Importantly, these data should somehow correlate/be discussed with respect to the D-loop assay performed in Fig. 1. The authors conclude that the binding to RNA is reduced in S97D-RAD51 suggesting that the pRAD51 that they observe at nuclear speckles would be probably not associated with RNA at these nuclear speckles, right? this goes against their idea of this phosphorylated form being related to RNA splicing...

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The manuscript seems to be in early days and requires lots of editing, rewriting to relate the in vitro and in cell data and make a coherent story

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    The authors performed chromatin fractionation to determine the correct localization of the pS97-RAD51 and looked for the phosphorylated form by western blots. But then they confirmed the finding using immunofluorescence. I think it would be more convincing and consistent if the authors do a pre-extraction before the use the antibody because as such, they would be indeed confirming the localization of the protein they are looking at that is specifically in the nucleus.

    As well, in order to test the specificity of the pS97-RAD51 antibody they generated, a simple treatment of the lysates with phosphatases would be a good control for the specificity of their antibody These and the critics mentioned above need to be address.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    This manuscript is not ready for submission

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes. However, the legends of the images are way too concise.

    • Are the experiments adequately replicated and statistical analysis adequate?

    In Fig. 2B, the authors performed a double thymidine block followed by a time course release to track cell cycle progression of the cells and phosphorylation of RAD51 at S97. They do not indicate the biological replicates they performed. There are no error bars in the estimated KD shown in Fig.7.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    The authors conclude that the S97 is specifically phosphorylated by the Aurora A kinase. How? Have they looked at other documented kinases known to phosphorylate RAD51?

    In figure 6 the authors overexpress HA-tagged RAD51 proteins corresponding to WT, S97D and S97A mutants in cells and label them for immunofluorescence. Maybe it would be better to downregulate the endogenous RAD51 to discard possible combined effects.

    In figure

    • Are prior studies referenced appropriately?

    The authors show in their manuscript that RAD51 protein CAN interact with RNA in vitro, a finding not previously described to my knowledge. However, a recent study entitled "RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops, Nature, 2020" provides in vitro data showing the binding of RAD51 to TERRA, a LncRNA, which I think would be worth mentioning their manuscript.

    The authors should mention previous contributions in the field especially when it comes to RAD51 in the HR pathway post DNA damage, which is quite documented and updated. For example, in this section of the introduction, "RAD51 is a recombinase protein implicated in the strand exchange mechanism during the DSB repair by the Homologous Recombination (HR) pathway. In the absence of DNA Damage (DD), RAD51 is predominantly cytoplasmic and translocates to the nucleus during the DNA Damage Response (DDR) to manage HR repair. As it needs the undamaged sister chromatid as a template, the HR repair pathway occurs mainly in the late S, G2 phases of the cell cycle. However, it has been documented that HR repair can also occur during G1 and early S phases, and in this case, the undamaged template used for the repair could be the homologous chromosome or an RNA transcript2". This statement is definitely worth more references.

    The same problem is recurrent in the rest of the introduction; therefore, it needs to be updated and better referenced.

    • Are the text and figures clear and accurate?

    The text needs a lot of editing to accurately describe the results, see for example: "The resulting KD evaluation shows that the S97D mutant had a dsDNA binding affinity lower to that of the WT (a KD of 2.26 μM for the S97D-RAD51 vs a KD of 0.38 μM for the WT RAD51). Concerning, the S97A mutant comparison to the WT RAD51, we observed modified association and dissociation curves that resulted in an identical affinity to dsDNA (a KD of 0.33 μM for the S97A-RAD51 vs a KD of 0.38 μM for the WT RAD51). We can conclude that in our in vitro conditions, the Ser97 phosphorylation has a high impact on RAD51 affinity for DNA by dividing its affinity by 5.8." Besides, the figures are of low quality and should be more carefully crafted and presented. Some experiments (such as the D-loop) are not represented in the figures.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Using a different representation for the graphs would be a plus (also see previous comments)

    Referees cross-commenting

    I think the other reviewers and I have raised very important and complementary points that will help the authors improve the quality of the manuscript substantially.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The discovery of a new phosphorylation site in RAD51 (S97) by Aurora A is potentially interesting for the field of the maintenance of genome stability as it could broaden the understanding of how such an important recombinase may be regulating the maintenance of genome integrity throughout the cell cycle. Also, the idea of RAD51 being involved in splicing and mRNA maturation seems very attractive and a very important conceptual advance. However, given the premature status of the text and the figures, the manuscript falls short to show convincing evidence.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    Many works are highlighting the role of RNA binding proteins as an integral part of the DNA damage response. In addition, a wealth of evidence in the literature suggest that many DNA repair proteins are RNA binding proteins, and that RNA is an important player in the DDR. The possible finding that RAD51 interacts with RNA and localize to nuclear speckles possibly acting in splicing is very interesting and attractive. How is Aurora A involved in this, what is the trigger, and whether RAD51 is binding RNA at these sites is still unclear.

    • State what audience might be interested in and influenced by the reported findings.

    Labs working in genome integrity mechanisms and the crosstalk between transcription and DNA repair would be interested.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view.

    Genome Instability, homologous recombination

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Homologous recombination is central to stabilizing the genome where Rad51 recombinase plays a pivotal role. Authors found unexpected localization of Rad51 to nuclear speckles. This localization is associated with a novel phosphorylation site of Rad51 at Ser97, which is phosphorylated by Aurora A. Because nuclear speckles are where RNA maturation occurs, authors argue for the possible involvement of Rad51 in modulating splicing, a previously unsuspected role for this important recombinase.

    Major points:

    1. The discoveries made in this paper heavily rely on the Phospho-S97 specific antibody (PS97 antibody). The biggest concern of this reviewer is that the validation of this material is not rigid enough. The specificity of this antibody against PS97 is validated only by PS97 peptide competition. The outcome is not convincing either; the PS97 signal seems quite resistant to Si-RAD51 in Fig2A. Furthermore, in Fig2B, PSer97 signal seems rather constant throughout the cell cycle while Rad51 signal fluctuates.

    These observations make this reviewer wonder if the authors are really detecting the phosphorylation of Rad51 with this material (i.e., PSer97 antibody).

    I suggest the authors validate this antibody by doing the following experiments:

    1-1. Do phosphatase treatment to see if the western blotting signal depends on phospho-S97.

    1-2. Do competition experiments using the non-phospho peptide (i.e., the same polypeptides carrying a regular unmodified Ser at 97).

    1-3. Try western blotting using purified Rad51 proteins, one treated with AuroraA and another without the treatment.

    1-4. Do western blotting with cell extract from the cell line producing Rad51-S97A, S97D and compare with wild-type Rad51.

    1. P.10, line 4 The purity of the purified protein should be included (Rad51 and two other mutant proteins) by showing CBB-stained SDS-page gel.
    2. P. 10, line 7 (Fig1C). D-loop assay with Rad51 and its mutants. The actual data should be presented with the actual D-loop formation efficiency. Comparison with wild type value is not enough.
    3. Fig5AB There are lots of PSer97 signals that do not even overlap with DAPI (Fig5A) or Sc35 (Fig5B). How do authors explain that? Also, quantification needs to be done regarding colocalization between PSer97 and Sc35.
    4. Fig5D I do not know what to look for here. At least authors should employ proper negative controls such as siRad51 extract and WCE supplemented with PSer peptides.
    5. Fig6AB Quantification of the results needs to be presented. This reviewer is wondering if there is any explanation regarding the difference in the localization of overproduced HA-Rad51 between HeLa and HCC1806; HA-Rad51 goes into a nucleus in HeLa while it stays in the cytoplasm in HCC1806. Any explanation?

    Minor points:

    1. Please include line numbers.
    2. P.2, line 11 Could you cite the literature showing Rad51 is predominantly cytoplasmic?
    3. P.10, line 15 The authors are not measuring the polymerization rate here. The title is misleading.
    4. Fig2A What do NT and Si-Sc stand for? How come Pser97 signal is resistant to Si-RAD51?
    5. Fig2B P-Rad51/Rad51 ratio graph does not have error bars, making it difficult to assess its reproducibility.

    Referees cross-commenting

    I am pretty much in agreement with the comments/criticisms raised by the other two reviewers.

    Significance

    If Rad51 is indeed involved in RNA maturation, that will be a very novel and exciting discovery. The observations presented in this work, however, seem a bit too inconclusive to support the idea, at least, to this reviewer.

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    Referee #1

    Evidence, reproducibility and clarity

    This paper by Alaouid et al. describes the role of Aurora A-mediated RAD51 phosphorylation in RNA metabolism in the sub-nuclear organelle such as Nuclear Speckles (NS). By the combination of biochemistry and cell biology methods, the authors showed that Aurora A phosphorylates Ser97 of human RAD51 in vitro. The antibody against the phosphor-Ser97 RAD51 seems to recognize NS. Moreover, RAD51 binds not only DNA, but also RNA in vitro. These suggest the role of RAD51 in RNA processing in NS. Moreover, the authors analyzed the biochemical properties of a phosphor-mimetic version of RAD51 (RAD51-S97D) and a phosphor-defective version (RAD51-S97A) and the effect of the over-expression of these mutants in NS dynamics in a cell. The idea of the link to RAD51 in RNA processing in a specific nuclear organelle sounds very interesting and opens a new area of DNA damage response (DDR). However, this paper did not show any functional evidence on the link between RAD51 phosphorylation and RNA processing in NS. Moreover, there are lots of technical issues in the results reported in the manuscript. In some experiments, the authors have to check the reproducibility and be careful about statistics. More importantly, the specificity of the anti-PSer97-RAD51 antibody raised in this study was not properly evaluated in vivo, which makes it hard to interpret the results using this antibody such as western blotting and immuno-fluorescent analyses.

    Major comments:

    1. Because of the poor description, it is hard to evaluate the content. This manuscript needs a more detailed description of the results in a scientifically valid way.
    2. Please describes the basic biochemical activities (ssDNA (Figure, dsDNA binding, and ATP hydrolysis activities) of mutant RAD51 proteins used in this study; RAD51-S97A and RAD51-S97D proteins. See also minor comments in this respect.
    3. It is essential to check the localization signal of PS97 signal in cells with RAD51 depletion by siRNA. Alternatively, the authors used chicken DT40 RAD51 cKO cells (Sonoda et al. EMBO J. 1998) to check the specificity of the antibody (RAD51 is conserved between humans and chicken, and the antibody seems to work in DT40).
    4. Data not shown: Please show the data in Supplemental Figures or deposit it in a public database.
    5. Please cite original references to cite the previous results.
    6. Please make a single composite Figure.

    Minor comments:

    1. Page 4, the second paragraph, line 1: Please add the reference number of Chabot et al. (4).
    2. Page 4, last sentence, DNA/RNA binding activity: For the binding of RAD51 in the presence of ATP, Mg2+ ion or divalent ion is essential. However, there is no description on how much concentration of the divalent ion was used in the assay.
    3. Page 10, the first paragraph, line 4: Please show a Coomassie gel of purified RAD51, RAD51-S97A, and RAD51-S97D proteins.
    4. Figure 1C, D-loop assay: Please show the gel of the products in this assay. It would be nice to show the kinetics of the reactions by these RAD51 mutant proteins. Or the effect of a different RAD51 concentration was tested.
    5. Page 10, the third paragraph, line 1: Please explain what is "BS3"; how this chemical stabilizes the oligomer and the references related to the drug.
    6. P values: Please describe the method to calculate the value in Figure legends.
    7. Figure 1D: Since this assay cannot quantitatively measure the oligomerization status of RAD51, the authors' claims are not convincing. Electron microscopic observation, which is the best, and/or ultra-centrifugation or gel filtration would be recommended to see the difference in the oligomeric status of the RAD51.
    8. Figure 2A: This result is not convincing. Although siRNA for RAD51 largely decreases the amount of RAD51 in cell lysates (bottom, ~80%), a modest decrease of the signal is seen for Phospho-Ser97-RAD51 (top, ~50%)). The authors need to explain this discrepancy. More importantly, this phosphorylation is mediated by Aurora A kinase. It is important to show the signals detected with this antibody decrease in the treatment of the Aurora A inhibitor or siRNA for Aurora A subunits. The inhibition experiments shown in Figure 4A are not convincing because the effect of the inhibitor is very small.
    9. Figure 2B: How did the authors determine each stage of G1, S, G2, and M phases (bottom right graph)? There are no markers of the S phase in western blots such as Cyclin A. Moreover, FACS analysis would be recommended.
    10. Figure 2B, graphs: Please add error bars of quantification of the bands by doing multiple experiments to support the authors' claim on the increase of RAD51 S97 phosphorylation from G1/S to G2/M transition.
    11. Figure 2C top, fractionation assay: Please include a western blot of RAD51 as a control like the ones in the middle.
    12. Figure 2D top: Please include images of RAD51 as a control.
    13. Page 12, first paragraph, line 2: Please show representative images of immunostaining of different cell lines in the Supplementary Figure and quantify the size of the foci. Do show all the data in either main Figures or Supplemental Figures without "data not shown".
    14. Figure 3A: Please explain gammaH2AX blot in either text or legend.
    15. Page 12, the second paragraph, line 8, data not shown: Please show the data in Supplemental Figure.
    16. Page 12, the third paragraph, line 3: The authors need to explain what is the gammaH2AX to readers.
    17. Figure 3B and C: Please check BRCA1/2 or RPA32 (or other DNA repair center markers) localization rather than gammaH2AX for the marker for DNA damage focus. As shown in these figures, gammaH2AX signals spread over the DSB sites, make it difficult to check the colocalization. Why number and intensity of gammaH2AX signals are so different between B and C? In Figure 3C, did the authors use non-treated cells?
    18. Figure 3B, western blots: The top panel is over-exposed.
    19. Page 12 last paragraph-page 13 first paragraph: The short summary is not necessary. These sentences should be moved to Discussion.
    20. Figure 4A: Please include any positive marker for the Aurora-A inhibition such as histone H3S10-phosphorylation.
    21. Figure 4B: Did Aurora A overexpression induce any cell cycle arrest? If it induces G2/M arrest, this increased phosphorylation is simply due to the arrest (in Figure 2B, the authors showed an increase of the phosphorylation in G2/M phase).
    22. Figure 4B pSer97-RAD51/RAD51 ratio: This reviewer is not convincing the quantification. What is the dynamic range of this western? Do they try different cell lysate volumes to adjust constant RAD51 signals to compare the pSer97-RAD51 signals?
    23. Page 13, third paragraph, lines 2-3 and Figure 4B left graph: Is this statistically significant? Please show what statistical method was used here (show it in Legend).
    24. Figure 5B, PlaB treatment: The Images show a decreased focus number of PSer97-RAD51. This is more obvious than the formation of larger foci. The authors need a more precise description of the result in the text.
    25. Figure 5C: Please show the position of the full-length of RAD51 protein by an arrow. The position of RAD51 and pS97 are different-pS97 signal migrates faster than the RAD51 (opposed to the result in Figure 1A).
    26. Figure 5D, IE: What is "NIP"?
    27. Figure 5D, IP: Where is a band of Sc-35? In IP fraction (bottom), there is little band corresponding with the band in lysates. Three thick band are not specific.
    28. Figure 5E, page 14 last sentence, "improved this experiment": Without the quantification, the authors do not conclude this.
    29. Figure 5 experiments: It is not clear why the improvement of Rad51-IP by RNA treatment could explain the role of pSer97-RAD51 points out the RAD51-binding to RNA. Rather the opposite interpretation would be possible. If pSer97-RAD51 is tightly bound to an RNA-containing nuclear structure, the authors may try chromatin fractionation with RNAase treatment.
    30. Figure 6: Please quantify the number and size of Nuclear speckles in different conditions.
    31. Figure label of B and D: "B" and "D" should be placed on the graph for RNA binding.
    32. Page 15-16, DNA/RNA binding assay: Please indicate the length of DNA/RNA in the text. Moreover, it is well established that ATP analogs modulate RAD51-binding to DNA. It is important for the authors to check the effect of ATP and ADP et on DNA/RNA.
    33. Page 16, the second paragraph: In this paragraph, the authors mentioned about "ds"DNA rather than ssDNA described above. Which is true?
    34. Supplemental Figure 1: Please explain what the purple circle means. Moreover, how this result shows the phosphorylation of Ser97. The two spectra look very different. Do they have any other phosphorylation?

    Referees cross-commenting

    I also agree with the other two reviewers. My concern is that we need a re-review of the revised version. I am not familiar with how the Review Common works. Hope that the journal will take care of the re-reviewing after the authors address our concerns on this paper

    Significance

    This paper may offer a new idea in the biology of nuclei by providing a possible link between proteins involved in homologous recombination such as RAD51 and RNA processing in subnuclear compartments, which is regulated by Aurora A-phosphorylation.

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. This paper might provide a possible link of RAD51 protein involved in homologous recombination with RNA processing in subcompartments in nuclei.
    • Place the work in the context of the existing literature (provide references, where appropriate). The concept on the role of RAD51 in nuclear RNA processing sounds interesting.
    • State what the audience might be interested in and influenced by the reported findings. The results in the paper are of interest to researchers who work on DNA damage response and DNA repair as well as RNA metabolism.
    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. I am a researcher on DNA damage response and DNA repair but is not working of RNA biology.
  5. Version published to 10.1101/2023.08.11.552966v1 on bioRxiv