The DNA molecule in our cells’ nucleus forms the instruction manual for proper cellular functioning. Unfortunately, the integrity of our DNA is continuously challenged by a variety of endogenous and exogenous agents (e.g., ultraviolet light [UV], cigarette smoke, environmental pollution, oxidative damage, etc.). These DNA lesions interfere with DNA replication, transcription, and cell cycle progression, leading to mutations and cell death, which may cause cancer, inherited diseases, or aging (Chatterjee and Walker, 2017).

To prevent the deleterious consequences of persisting DNA lesions, all organisms are equipped with a network of efficient DNA repair systems. One of these systems is the Nucleotide Excision Repair (NER) which removes helix-distorting DNA adducts caused by UV such as Cyclo-Pyrimidine Dimers (CPDs) and 6-4 Photoproducts (6-4PPs) (Giglia-Mari et al., 2011).

In mammals, the different steps of NER require more than 30 different proteins that are recruited sequentially to the DNA damage site, as demonstrated by the different studies of NER proteins kinetics (Moné et al., 2004; Politi et al., 2005; Rademakers et al., 2003; van den Boom et al., 2004; Zotter et al., 2006). The first step of NER consists of damage recognition, followed by the opening of the DNA duplex, dual incisions on both sides of the damage, excision of 24–32 oligonucleotides containing the damage and, finally, gap-filling by repair DNA synthesis.

NER is divided into two subpathways depending on DNA lesions position within the genome. The Global Genome repair (GG-NER) will detect and repair lesions throughout the genome, whereas Transcription-Coupled repair (TC-NER) is associated with RNA polymerase 2 (RNAP2) to repair lesions on the transcribed strand of active genes (Marteijn et al., 2014).

The NER system has been linked to rare human diseases, classically grouped into three distinct NER-related syndromes. These include the highly cancer-prone disorder Xeroderma Pigmentosum (XP) and the two progeroid diseases: Cockayne Syndrome (CS) and Trichothiodystrophy (TTD). Importantly, CS and TTD patients are not cancer prone but present severe neurological and developmental features (Hanawalt, 1994).

Xeroderma Pigmentosum group A (XPA)-binding protein 2 (XAB2) is a highly conserved protein of 100 kDa and consists of 15 tetratricopeptide repeat (TPR) motifs that carry out protein–protein interactions. XAB2 protein was identified as a protein interacting with XPA, a NER factor, using a yeast two-hybrid system (Nakatsu et al., 2000). Next, it has been shown that XAB2 interacts also with the TC-NER-specific factors, CSA and CSB, and the elongating form of RNAP2 (Nakatsu et al., 2000). XAB2 is also essential for early mouse embryogenesis, as demonstrated by the preimplantation lethality observed in XAB2 knockout mice (Yonemasu et al., 2005).

Downregulation of XAB2, using either anti-XAB2 or siRNA, inhibits normal RNA synthesis and the recovery of RNA synthesis after UV irradiation (Kuraoka et al., 2008; Nakatsu et al., 2000). Furthermore, injection of anti-XAB2 in GG-NER-deficient cells significantly reduces UV-induced Unscheduled DNA Synthesis (UDS) during repair (Nakatsu et al., 2000). These results suggest the involvement of XAB2 in transcription and TC-NER.

Further studies have shown that XAB2 is a component of the Prp19/XAB2 complex (Aquarius [AQR], XAB2, Prp19, CCDC16, hISY1, and PPIE) or Prp19/CDC5L-related complex required for pre-mRNA splicing (Kuraoka et al., 2008). XAB2, as well as PRP19 and AQR, has been involved in the DNA damage response (DDR) (Maréchal et al., 2014; Onyango et al., 2016; Sakasai et al., 2017). Indeed, XAB2 is essential for homologous recombination (HR) by promoting the end resection step (Onyango et al., 2016). PRP19 is a sensor of RPA-ssDNA after DNA damage (Maréchal et al., 2014) and AQR contributes to the maintenance of genomic stability via regulation of HR (Sakasai et al., 2017).

Interestingly, AQR has a role in removing R-loops, a three-stranded nucleic acids structure composed of a DNA:RNA hybrid and the associated nontemplate single-stranded DNA (Sollier et al., 2014). These structures can form during transcription, when an RNA molecule emerging from the transcription machinery hybridizes with its DNA template. They are found abundantly in human gene promoters and terminators where RNA processing occurs (Wang et al., 2018).

Despite the knowledge acquired in the last decades on XAB2 and its different cellular roles, little is known about the exact crosstalk and dynamics between its diverse cellular functions, specifically between DNA repair transcription and splicing. In this work, we describe the molecular dynamics of XAB2 within the cell after UV-damage induction and during the TC-NER repair process. We determined in vivo that, in the absence of XAB2, Transcription-Coupled repair reactions are impaired, consequently, restart of transcription after UV damage is abolished. Surprisingly, unlike all the other NER proteins studied so far, the mobility of XAB2 is increased after irradiation, and XAB2 shows no accumulation on local UV lesions. This changing dynamic is not restored until DNA repair is completed. Indeed, in damaged TC-NER-deficient cells, XAB2 remains more mobile. Interestingly, we demonstrate that, after DNA damage induction, XAB2 is not released from the splicing complex but is detached from R-loops, a recently XAB2 identified substrate (Goulielmaki et al., 2021). Additionally, we investigate the relation between XAB2 and RNAP2, demonstrating that XAB2 retains RNAP2 on its substrate. Moreover, in the absence of XAB2, RNAP2 interacts strongly and durably with both types of UV lesions (6-4PPs and CPDs), suggesting a role of XAB2 in the DNA damage recognition step of TC-NER.

Helix-distorting lesions continuously challenge cell survival by interfering with and blocking fundamental cellular functions, such as transcription and replication. In order to prevent the deleterious effects of these events, cells have developed different mechanisms to restore an undamaged DNA molecule and allow the restart of cellular processes. The importance of rapidly re-establishing perturbed cellular functions is underlined by the presence of a repair mechanism directly coupled with transcription, like the TC-NER.

Two phases can be distinguished during TC-NER events: (1) the actual repair reaction of the damaged strand via the TC-NER subpathway and (2) the resumption of transcription after repair. The experiment known as ‘RRS’ measures only the restart of transcription after DNA repair completion and thus the involvement of a protein in the general TC-NER process. However, this assay does not discriminate between the two phases of the TC-NER. As a consequence, proteins that are fully proficient in the repair reaction but fail in the transcription restart after DNA repair completion might have the same defect in RRS as those solely deficient in the DNA repair reaction.

Thus far, all studies have demonstrated the involvement of XAB2 in the TC-NER process using only RRS experiments (Kuraoka et al., 2008; Nakatsu et al., 2000). In this study, we used a specific test developed in-house (Mourgues et al., 2013) called TCR-UDS and thus demonstrated that, indeed, XAB2 is solely needed for the repair reaction (Figure 1D). This is confirmed by PLA experiments showing an interaction between XAB2 and helix-distorting lesions, 6-4PPs and CPDs, after irradiation (Figure 2 and Figure 2—figure supplement 1). However, these experiments do not clearly discriminate at which step of the repair reaction XAB2 may contribute. Two hypotheses are envisaged: (1) XAB2 functions in damage recognition or (2) in the repair reaction per se.

XAB2 has been found as part of the pre-mRNA splicing complex composed of AQR, PRP19, CCDC16, PPIE, and ISY1 (Kuraoka et al., 2008). However, none of these proteins take part in the repair process, underlying the peculiar function of the splicing factor XAB2 in TC-NER repair (Figure 3—figure supplements 1–3).

We also demonstrated that, unlike all the other NER proteins studied so far, XAB2 protein is released from the damaged region induced by UV-C exposure (Figure 3). Concomitantly, we also observed an increase in XAB2 cellular mobile fraction after UV irradiation (Figure 4A). This release from DNA-damaged area and increased mobility is surprising and atypical for a repair protein. However, this behavior has also been observed for late-stage spliceosomes (Tresini et al., 2015) and could be explained by the importance of the cell rapidly providing access to the repair machinery.

Surprisingly, the increased XAB2 mobility occurs in the absence of CSA and CSB proteins, with inhibitors of DDR after UV irradiation but also after transcription inhibition (Figure 4 and Figure 4—figure supplement 1). These results strongly suggest that XAB2 remobilization is independent of the repair process but it is more a result of the transcription inhibition induced by the DNA damage. Moreover, the recovery of intrinsic XAB2 mobility is CSA and CSB dependent probably because in TC-NER defective cells, repair of transcribed genes is deficient and transcription is not recovered. As a consequence, a proper repair and re-establishment of the transcription process are needed to restore XAB2 mobility to normal values.

Previously, we reported that a complete TC-NER mechanism is required to repair the UV lesions present on active rDNA, genes transcribed by RNAP1 (Daniel et al., 2018). Notably, both Cockayne syndrome proteins (CSA and CSB) are implicated in this specific repair reaction, as well as the UV-stimulated scaffold protein A (UVSSA), a protein required for the stabilization of CSB specifically after UV irradiation (Higa et al., 2016). By measuring the level of ribosomal RNA, we demonstrate that RNAP1 transcription restarts after irradiation in the absence of XAB2 (Figure 2—figure supplement 2), meaning that UV lesions present on active rDNA are repaired. As a consequence, XAB2 is involved only in TC-NER of RNAP2-transcribed genes and probably not in the repair of RNAP1-transcribed genes, confirming a likely specific interaction with RNAP2 and reinforcing the idea that RNAP1 and RNAP2 repair processes are distinct although they share common proteins.

FRAP experiments in CSA and CSB mutant cells show a more immobile XAB2 fraction than in WT cells (Figure 4B) without any damage induction. Tanaka’s group found that XAB2 interacts in vitro with CSA and CSB protein in the absence of DNA damage (Nakatsu et al., 2000). In addition, several studies demonstrated the involvement of CSB in transcription regulation (Boetefuer et al., 2018). As both CSB and XAB2 are necessary during the transcription process, it is therefore possible that the absence of CSB will modify XAB2 mobility. However, it is not excluded that in CSA and CSB mutant cells, a low level of unrepaired oxidative damage (de Waard et al., 2004) might interfere with the proper XAB2 mobility, eventually modifying the amount of R-loops within these cells. Nevertheless, it is difficult to precisely estimate the exact number of R-loops between different cell types, and this hypothesis is difficult to clearly assess.

The UV-induced remobilization of XAB2 is not explained by its release from the splicing complex, as demonstrated by co-immunoprecipitation and PLA experiments, but it is more the result of the release from chromatin-specific structures, in this particular case the R-loops (Figure 5). An R-loop is a three-stranded nucleic acid structure composed of a DNA:RNA hybrid and the associated nontemplate single-stranded DNA. This structure arises naturally in organisms from bacteria to humans, and has a multitude of functions in the cell (Belotserkovskii et al., 2018). Our results show that R-loops are a substrate for XAB2, and after DNA damage induction, the interaction between XAB2 and R-loops is strongly reduced. This might explain the increased XAB2 mobility during the TC-NER reaction.

It was previously demonstrated that, in AQR-depleted cells, R-loops formation is induced. These R-loops are actively processed into DNA double-strand breaks by XPF and XPG, the NER endonucleases (Sollier et al., 2014). Without any damage, we also observed an increased level of cellular R-loops in both AQR- and XAB2-silenced cells (Figure 5C), suggesting an involvement of these two proteins in R-loops resolution, as recently also demonstrated by Goulielmaki et al., 2021.

Transcription process and R-loops formation are finely interconnected. Indeed, R-loops formation can cause transcription blockage. Transcription blockage due to DNA damage appears to result in R-loops formation (Mullenders, 2015; Steurer and Marteijn, 2017). Moreover, transcription activity declines after UV irradiation and mRNAs levels are drastically reduced (Figure 5—figure supplement 5). PLA assays show that, after UV irradiation, XAB2 and total RNAs interaction is reduced. However, because the total amount of RNAs is diminished after transcription block, we assume that XAB2_RNAs interaction is lessened because of the intrinsic reduced amount of RNAs. Differently from total RNAs, in our study, we observe that R-loops do not decrease in number after UV damage and transcription inhibition (Figure 5E). Therefore, the interaction XAB2_R-loops and AQR_R-loops is specifically hindered after UV damage (Figure 5D). However, a careful interpretation of these results should be made because a recent paper demonstrates that the S9.6 antibody, specific for DNA:RNA hybrids, can also recognize other nucleic acid structures in immunofluorescence assay (Smolka et al., 2021). To be able to confirm our results, many additional controls were performed: (1) removal of the cytoplasm before fixation (Figure 5—figure supplement 2A); (2) subtraction of the nucleolar signal from the nuclear signal (Figure 5—figure supplement 2B); (3) treatment with RNAseH which degrade specifically R-loops (Figure 5—figure supplement 4); and (4) finally interaction of XAB2 with nascent RNA (EU staining) is different from interaction with R-loops (Figure 5—figure supplement 5). Although all controls point to a seeming interaction between XAB2 and R-loops or between AQR and R-loops, we cannot exclude that XAB2 and AQR do not release from DNA:RNA hybrids but from other nucleic acid structures, for example, double-stranded DNA.

Because XAB2 interacts in vitro with CSA and CSB protein (Nakatsu et al., 2000) and recent studies have shown an interaction with XPG (Goulielmaki et al., 2021), we decided to verify whether these interactions were modified during the DNA repair process and the concomitant transcription inhibition. Our results show a clear and consistent reduction of interaction between XAB2 and CSA (Figure 6A, B) and between XAB2 and XPG (Figure 6C, D) but not between XAB2 and CSB or between XAB2 and XPB (Figure 6—figure supplement 1). These results suggest that XAB2 intervenes in the early steps of TC-NER or at least in the steps that imply the activity of CSB and TFIIH and most probably the early RNAP2 blocking-recognition step. However, the reduction of interactions between XAB2 and CSA or XPG is less striking than the one with R-loops. As suggested in Goulielmaki et al., 2021, another working hypothesis would be that XAB2 is released from CSA and XPG proteins associated with R-loops processing.

The physical interaction between XAB2 and RNAP2 has already been established (Kuraoka et al., 2008; Nakatsu et al., 2000), but the exact relation between XAB2 and RNAP2 has not yet been disclosed. Without DNA damage, we observed a difference in nascent RNA synthesis in XAB2-silenced cells compared to control cells (Figure 1—figure supplement 2B). Since XAB2 functions in both transcription and splicing, it is not surprising that the quantity of nascent RNA is altered in the absence of XAB2 (Goulielmaki et al., 2021; Kuraoka et al., 2008). Moreover, we have clearly demonstrated by FRAP experiments that RNAP2 mobility is severely affected in the absence of XAB2. Namely, in XAB2-depleted cells, RNAP2 is released from its substrate and its mobility is strongly increased (Figure 7A). RNAP2 immobile fraction after UV irradiation does not change significantly in the presence or absence of XAB2. However, we could observe that interactions of RNAP2 with the UV lesions (6-4PPs or CPDs) are stronger after DNA damage and last longer in siXAB2-treated cells compared to siMock-treated cells (Figure 7B–D). These results suggest that, during transcription, XAB2 helps RNAP2 anchoring to its substrate, whereas during the TC-NER process, it has an effect on stalling of RNAP2 on UV lesions, advocating for a potential role in the DNA damage recognition step.

In conclusion, we describe here an increased mobility of the protein XAB2 during the DNA damage-dependent transcription inhibition. This increased mobility might partly be explained by the release of XAB2 from its substrate R-loops and its partner CSA and XPG. Importantly, we demonstrate that XAB2 plays an anchoring role for RNAP2 to its substrate during transcription and helps RNAP2 to detach from UV lesions after DNA damage. As aconsequence, the absence of XAB2 hinders the overall transcription activity of the cells and severely affects the TC-NER capacity (Figure 8).

Figure 8

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All data generated or analyzed during this study are included in the manuscript and supporting file.

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The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The manuscript is suited for eLife and the revision plan as presented by the authors is sound. Here is a list of essential revisions which includes the planned revisions by the authors and some additional points added by the reviewing editor. This implies agreement with the author's choice of revisions NOT to be done but see point #5.

Author list

1) Further PLA experiments with RNAP2 and antibodies against CPD and 6-4PP in the presence and absence of UV and/or siXAB2 will strengthen the point about RNAP2 mobility.

2) Resolve the conflict with Nakatsu et al. 2000 and Kuruoka et al. 2008 regarding the reported increase in transcription with RNAseq experiments with siXAB2 +/- UV. This may resolve the conflict or may not, see #5.

3) Benzonase treatment in IP XAB2 experiments represents a critical control.

4) The plans for the expanded discussion and the changes already implemented are fine.

Editor list

5) Regardless of the outcome of point #2, this conflict will need to be resolved, which may need additional experimentation, if the outcome in inconclusive or cannot explain the discrepancy.

6) I agree that additional experimentation regarding RNAP1 is not needed in the revision, but the conclusion will have to be significantly toned down and limitations be discussed regarding the claimed RNAP2 specificity.

7) The authors should tone down the claim that S9.6 is specific for R-loops, the antibody recognizes also other nucleic acid structures. See J.A. Smolka, L.A. Sanz, S.R. Hartono, F. Chedin Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids J. Cell Biol., 220 (2021).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "XAB2 Dynamics during DNA Damage-Dependent Transcription Inhibition" for further consideration by eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The revision addresses the essential points in the previous review. The manuscript requires extensive language editing. There are numerous problems already in the abstract (line 4 'demonstrate', line 7 'becomes', line 8 'change in mobility is restored' needs rephrasing, line 10 'from DNA:RNA hybrids', line 11 'proteins', line 13 'demonstrate').

Reviewer #4 (Recommendations for the authors):

The addition of PLA data for the interaction of both RNAP and XAB2 with UV photoproducts is very welcome, and the data quite clearly show increased interaction with damage for both proteins. However, these results actually demand some rethinking of the conclusions.

The authors addressed the essential points for the revision but the manuscript requires extensive language editing.

[Editors' note: this paper was reviewed by Review Commons.]

Essential revisions:

The manuscript is suited for eLife and the revision plan as presented by the authors is sound. Here is a list of essential revisions which includes the planned revisions by the authors and some additional points added by the reviewing editor. This implies agreement with the author's choice of revisions NOT to be done but see point #5.

Author list

1) Further PLA experiments with RNAP2 and antibodies against CPD and 6-4PP in the presence and absence of UV and/or siXAB2 will strengthen the point about RNAP2 mobility.

This PLA experiment has been performed and we observed more interactions between RNAP2 and UV lesions, 6-4PPs and CPDs, in siXAB2-treated cells compared to siMock-treated cells. Moreover, these interactions last longer in time. These results are described in the last part of the “Results section” (lines 539-547). Graphs with signal quantification have been included in Figure 7 together with the result of RNAP2 dynamic, measured by FRAP. We also added representative images of this experiment in Figure 7 —figure supplement 1.

In parallel, because the results of these PLA “RNAP2-UV-lesions” revealed that depletion of XAB2 influence (directly or indirectly) the retention of RNAP2 on UV-lesions, we decided to perform a PLA experiment between XAB2 and UV lesions after irradiation. The results obtained show that XAB2 interacts directly with or is in proximity of 6-4PP and CPD lesions until their removal and because this is the first time that such a result is obtained, we tool the initiative to add these results in the manuscript in Figure 2 and figure supplement 2. To describe these results, new text has been implemented in the “Results section”, lines 121-127.

2) Resolve the conflict with Nakatsu et al. 2000 and Kuruoka et al. 2008 regarding the reported increase in transcription with RNAseq experiments with siXAB2 +/- UV. This may resolve the conflict or may not, see #5.

Editor list

5) Regardless of the outcome of point #2, this conflict will need to be resolved, which may need additional experimentation, if the outcome in inconclusive or cannot explain the discrepancy.

Unfortunately, the RNAseq experiments to resolve the conflict with Nakatsu at al. 2000 and Kuruoka et al. 2008 could not be performed because this technique finally requires too much development for our lab at the moment.

Nevertheless, we could resolve the conflict with Nakatsu at al. 2000 by changing the time of EU incubation. Indeed, with 1 hour of EU incubation (the same time frame used in the work of Nakatsu) the quantity of nascent RNA is reduced in absence of XAB2 compared to control cells. The difference of nascent RNA synthesis between 1h and 2h of EU incubation could be explained by the role of XAB2 in transcription as well as in splicing. New text has been implemented in the “Results section”, lines 102-107.

We decide not to explore further this result because we consider it is not in the main scope of this article and it does not change our conclusions.

3) Benzonase treatment in IP XAB2 experiments represents a critical control.

As suggested by the reviewers #4, we performed Benzonase treatment to confirm the interaction of XAB2 with R-loops. We observed that after benzonase treatment and immunoprecipitation with S9.6 antibody, XAB2 is not anymore revealed by western blot, confirming the interaction of XAB2 with DNA:RNA hybrids. The figure and the text corresponding to this part of result have been changed, lines 390-394 and Figure 5B.

4) The plans for the expanded discussion and the changes already implemented are fine.

6) I agree that additional experimentation regarding RNAP1 is not needed in the revision, but the conclusion will have to be significantly toned down and limitations be discussed regarding the claimed RNAP2 specificity.

We changed/added text in the ‘Discussion section’ according to the new results:

Moreover, we tone down our conclusion about the specificity of XAB2 to repair only RNAP2 genes (line 604-607)

7) The authors should tone down the claim that S9.6 is specific for R-loops, the antibody recognizes also other nucleic acid structures. See J.A. Smolka, L.A. Sanz, S.R. Hartono, F. Chedin Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids J. Cell Biol., 220 (2021).

In the paper of Smolka et al. 2021, it has been demonstrated that S9.6 signal is prominently cytosplasmic and nucleolar and derive mainly from ribosomal RNA. It was not well described in our manuscript in the first version, but when we performed immunofluorescence with S9.6 antibody, we removed the cytoplasm before fixation and; during quantification, the nucleolar signal was subtracted from the nuclear signal. This information was described in Figure 5 —figure supplement 2 and new text has been implemented in the ‘Results section’, line 395-400.

A new paragraph (line 644-654) was added in the ‘Discussion section’ to precise that although a lot of controls have been performed to confirm the specificity of the interaction of XAB2 and AQR with R-loops, we cannot exclude that these proteins release from other nucleic acid structure, for example double-stranded DNA.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The revision addresses the essential points in the previous review. The manuscript requires extensive language editing. There are numerous problems already in the abstract (line 4 'demonstrate', line 7 'becomes', line 8 'change in mobility is restored' needs rephrasing, line 10 'from DNA:RNA hybrids', line 11 'proteins', line 13 'demonstrate').

Reviewer #4 (Recommendations for the authors):

The addition of PLA data for the interaction of both RNAP and XAB2 with UV photoproducts is very welcome, and the data quite clearly show increased interaction with damage for both proteins. However, these results actually demand some rethinking of the conclusions.

The authors addressed the essential points for the revision but the manuscript requires extensive language editing.

We would like to thank the referees for having reviewed positively our manuscript for ‘ELife’. As requested, we tried to make extensive language editing but we are not English speaking. The new uploaded Article File include tracked changes indicating the revisions made, using the tracked changes function in Word. The following texts have been extensively changed:

Results part – XAB2 dynamic during TC-NER – 1^st paragraph.

Old version:

“In this technique, fluorescence molecules are photo-bleached in a small spot by a high intensity laser pulse; then the recovery of fluorescence within the bleached area is monitored over time. With no treatment, this measure of fluorescence recovery corresponds to the protein mobility within the living cells. After perturbation of the nuclear environment (e.g. DNA damage), a protein can become less mobile if it physically interacts with a new substrate or a bigger complex; more mobile if the protein is released from its substrate; or eventually will not change its mobility.”

New version:

“In this technique, fluorescence molecules are photo-bleached in a small spot by a high-intensity laser pulse. Subsequently, fluorescence recovery within the bleached area is monitored over time. When cells are untreated, the measure of fluorescence recovery corresponds to the protein intrinsic mobility within the living cells. After perturbation of the nuclear environment (e.g., DNA damage), a protein can physically interacts with a new substrate or a slower complex, becoming less mobile or on the contrary can be released from its substrate, becoming more mobile. Eventually, the protein can also have an unchanged mobility.”

Results part – XAB2 is not released from the splicing complex during DNA repair reactions – 2^nd paragraph.

Old version:

“In order to distinguish between these two possibilities, we firstly investigated whether XAB2 dissociates after DNA damage induction from the splicing complex described earlier. XAB2 was immunoprecipitated together with AQR. Interestingly, XAB2 was strongly and consistently immunoprecipitated 1-hour post-irradiation which corresponded to the time in which XAB2 mobility is increased and at the same time point more AQR is also immunoprecipitated. No clear release of XAB2 from AQR was observed at different time points.”

New version:

“In order to distinguish between these two possibilities, we firstly investigated whether, after DNA damage induction, XAB2 dissociates from its splicing partner AQR by immunoprecipitating XAB2 and AQR. Interestingly, XAB2 was immunoprecipitated more strongly and consistently 1-hour post-irradiation, time that corresponds to the XAB2 mobility increase. At the same time point, more AQR is also immunoprecipitated. No clear release of XAB2 from AQR was observed at different time points.”

Results part – XAB2 is released from R-loops during DNA repair reactions – End of this part.

Old version:

“Next, we verify whether XAB2 increase in mobility after UV irradiation is due to a decreased interaction with messenger RNA (mRNA). A decrease in the interaction between XAB2 and mRNA was observed but this decrease corresponded to the decrease of mRNA caused by UV-dependent transcription inhibition invalidating the results observed in the PLA XAB2-mRNAs.”

New version:

“Next, we verified whether the increase in XAB2 mobility observed after UV irradiation is due to a decreased interaction with messenger RNA (mRNA). PLA results show that a reduction in the interaction between XAB2 and mRNA was observed. However, this reduction paralleled the decrease of mRNA caused by UV-dependent transcription inhibition, suggesting that the results observed in the PLA XAB2-mRNAs are due mainly to a decrease in mRNAs amount.”

[Editors' note: this paper was reviewed by Review Commons.]

General Statements

We would like to thank the referees for having reviewed our manuscript. Their comments and requests will help us to improve our manuscript.

Description of the planned revisions

Reviewer 3 thinks that the enhanced mobility of RNA polymerase 2 (RNAP2) after UV due to the absence of XAB2 might be a central observation that should be further explored. As suggest by the reviewer, we will perform PLA with the RNAP2 and CPD or 6-4PP antibody with or without UV and/or siXAB2 to observe if stalling RNAP2 might be affected.

Our figure S2B is in direct contrast with published findings (Nakatsu et al., 2000; Kuraoka et al. 2008) and with our FRAP experiment on RNAP2 as mentioned by reviewer 4 (point 1 and 2) and also reviewer 1. Indeed, we observed by fluorescence of EU incorporation more RNA in siXAB2 treated cells compared to siMock treated cells, without any damage. An increase of RNA is frequently correlated with an increase in transcription. However, as XAB2 is a splicing factor, we could hypothesize that the observed increased RNA may be linked to splicing defect. Indeed, the paper of Goulielmaki et al. 2021 already show by RNAseq analysis a global impairment of splicing machinery in siXAB2 cells. Nonetheless, this assay was performed with total RNA without any damage. We propose to repeat this experiment of RNAseq in siMock and siXAB2 cells on nascent RNA and in cells UV-irradiated or not.

We will repeat the IP XAB2 experiment with benzonase digestion as propose by reviewer 4 (point 5). Benzonase remove nucleic acid and by consequence R-loops. The experiment will probably explain why the amount of immunoprecipitated XAB2 increased after UV.

The discussion will be changed according to the new result and to take into account all the comments of reviewer (for example point 3, 6, 7 and 9 of reviewer 4). A hypothesis will also be proposed. The numerous typographical, grammatical and language errors throughout the manuscript noted by reviewer 4 will be corrected at the end.

Description of the revisions that have already been incorporated in the transferred manuscript

We have already carry out the following minors modifications in the transferred manuscript as suggest by reviewer 2, 3 and 4.

Description of analyses that authors prefer not to carry out

Reviewer 1 suggest to present the quantification results of PLA as foci count rather than signal intensity. This method is not applicable in our PLA because with obtain many foci which are difficult to distinguish. Moreover, there is a direct correlation between the number of foci and the signal intensity.

Reviewer 2 ask to include in Figure 2 images of baseline (NoUV). This request is not necessary as the fluorescence in the local damage is compared to the fluorescence of the whole nucleus without local damages and without nucleolus.

Reviewer 4 (point 4) find our RNAFish 47S not strong enough to conclude that XAB2 is not involved in TCR of RNAP1-transcribed gene because the recovery in WT cells is higher (150%) than the starting value (without irradiation, 100%), whereas in siXAB2 cells it returns exactly to pre-damage level. Usually, in WT cells the recovery is similar to non-irradiated condition but like any living organism the results are never completely reproducible. In conclusion, we will not perform further experiment to strengthen our RNAFIsh 47S result.

Reviewer 2 ask why we used a student’s test while data are presented with 3 or more groups, for which this test is not valid. We will not change our statistic method as we always compared only two different conditions: after irradiation compared to No UV condition or siRNA X compared to siMock. It was not specified in our manuscript and we correct this forgets in the figure legends and in the methods section.

Reviewer 2 and reviewer 4 would like better western blot pictures. We already put in the manuscript our best representative western blot.

Author details

1. Lise-Marie Donnio

   Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Elena Cerutti

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2414-6034

2. Elena Cerutti

   Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Methodology, Writing – original draft

   Contributed equally with

   Lise-Marie Donnio

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4644-4817

3. Charlene Magnani

   Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Damien Neuillet

   Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Pierre-Olivier Mari

   Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France

   Contribution

   Data curation, Software, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

6. Giuseppina Giglia-Mari

   Institut NeuroMyogène (INMG), CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   ambra.mari@univ-lyon1.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2001-1965

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