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    Referee #3

    Evidence, reproducibility and clarity

    In this paper by the Vagner lab, co-transcriptional cleavage (CoTC) is discovered as a mechanism to ensure 3'-end processing of selected genes under conditions of global inhibition of 3'-end processing under DNA damage conditions.

    While global inhibition of 3'-end processing acts as a fail-safe mechanism to ensure that mutations arising from DNA damage are not propagated (and instead repaired), the expression of repair genes must be maintained under these conditions. In an elegant serious of experiments, Sfaxi and coauthors have identified CoTC as a mechanism, which allows specific pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.

    This is an important discovery, the article reads well and most of the experiments are technically sound. That having said, I think the authors did not 'sell' this important finding very well. While they initially started their studies based on processing of the p53 pre-mRNA, they later performed RNAseq (Figure 5&6) to identify further genes that are regulated in a CoTC-dependent manner - analogous to the p53 pre-mRNA. In doing so, the authors identify and validate further CoTC-dependent genes. Given the global RNAseq approach that the authors have undertaken but not yet fully exhausted, there are probably more genes hidden that are processed in a similar way.

    I would recommend harvesting the hidden treasure to more comprehensively understand CoTC-dependent processing and its relation to DNA damage conditions. In my opinion, it would be important to perform

    1. GO enrichment analyses of the 108 pre-mRNAs more effectively processed under UV and address the question whether DNA damage repair-related terms can be retrieved
    2. study of further DNA-damaging conditions to address the question if similar patterns and genes can be retrieved under these conditions
    3. complementary analysis of data derived from tumor genome databases whether mutations in the identified CoTC-sites are enriched (which one would expect)

    Finally I wonder, whether a small scheme illustrating conventional versus CoTC processing could enhance access for a broader readership.

    Minor Comments:

    Page 4, 2nd paragraph, last sentence: what is "vcxPAS"?

    Page 6, 2nd paragraph, the TBP RNA is not explained.

    Page 6, 2nd paragraph and following paragraphs. The PCF11-depletion experiments to sort out conventional versus CoTC-dependence is not very well controlled: it is surprising to see that depletion of PCF11, which is already absent under UV (Fig. 1A), seems to modulate processing of TBP under this condition (Fig. 1E). In order to turn this into a bona fide positive control for the entire experimental set-up, it is relevant to show that there are residual PCF11-levels under UV that can be further downregulated by siRNAs under this condition (currently this is not supported by the WB data in Fig. 1B).

    Page 8, 1st paragraph, last sentence: I find the reference to GAPDH and WDR13 as part of a figure that comes far below is a bit confusing

    Page 9, last paragraph, page 10, 1st paragraph: What does the analysis of WDR12, GAPDH and TBP exactly control for?

    Figure 3. Overall, I find the information shown in Fig. 3 somewhat confusing and wonder whether the quality can be improved (partial co-localization of spots, are the spots shown in D -UV an artifact? Etc.)

    Figure 4. I find the composition of panels C and D not very intuitive (I would reorganize the data such that each panel shows the RNA and protein expression data for each candidate individually (panel C for p53 and panel D for p21, respectively)

    Figure 5, panel B: The figure shows that the by far largest number of genes (>3000/3722) is not differentially regulated under UV compared to no-UV conditions. Does this question the commonly made statement that 3'-end processing is globally inhibited under UV quoted here (page 14, 2nd paragraph) and elsewhere?

    Referees cross-commenting

    @reviewer 1 & 3: I fully agree; the PCF11 depletion under UV-conditions is clearly visible. Thank you!

    Significance

    This is an important study to better understand the function and target genes of CoTC-mediated 3'-end processing. It thereby extends earlier studies mainly adressing the underlying molecular mechanisms and rationalizes the function (and evolution) of this gene regulatory principle.

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    Referee #2

    Evidence, reproducibility and clarity

    DNA damage leads to transient inhibition of 3' end processing and a marked reduction of cellular Pcf11 levels, a component of the cleavage factor II (CF II). In previous work, St¬ephan Vagner and colleagues described that p53 mRNA escapes the 3' end processing inhibition through the actions of the DHX36 RNA-helicase and the heterogenous RNA binding protein hnRNP H/F. However, this previous work failed to identify a mechanism through which DHX36 and hnRNP H/F would mediate the escape. In this work Stephane Vagner and his colleagues describe now how a sequence located 1200 nt downstream of the P53 poly(A) signal (PAS) is required to alleviate p53 mRNA from the general DNA-damage-dependent 3' end processing block. The presence of this element makes p53 mRNA 3' end processing independent from CTD-serine-2-phosphorylation and the CFII factor Pcf 11. As a high proportion of non-cleaved p53 pre-mRNA can be found in the nucleoplasm, the authors suggest that this element may be a co-transcriptional cleavage inducing site (CoTC). Deletion of this element renders p53 mRNA susceptible to DNA-damage-induced 3' end processing inhibition and leads to a p53 protein reduction after UV-exposure. Consequently, expression of down-stream targets of p53 is also reduced. The authors identify 9 more candidates for CoTC in other genes encoding for proteins required for the DNA-damage response.

    Major points:

    Although Figures 4 and S7 clearly show how long the RNA is that is retained on the chromatin, it is not clear, how long the RNA is that is released into the nucleoplasm. A Co-TC mechanism would pose that either nucleoplasmic CPSF-mediated cleavage and/or nucleoplasmic exonucleolytic degradation preceded nucleoplasmic polyadenylation. Neither of these points has been addressed by the current manuscript. This could be addressed by transcript reverse transcription analysis with nuclear RNA, in the background of an exosome ko to see if this would allow stabilisation of the un-cleaved RNA and its detection in the nucleoplasmic fraction.

    Minor points:

    It would be good if the site of action of DHX36 and hnRNP H/F could be repeated and put into context with the Co-TC element.

    Introduction; I feel the pause-type termination is probably better explained in either Cortazar et al 2019 or Gromak et al 2006.

    There may be a typo towards the end of the second paragraph: vcxPAS. If this is not a typo, it would be helpful to have this explained better.

    Figure 1A/B the conclusion: "These observations suggest that PCF11 might be dispensable for pre-mRNA 3' end processing following UV-induced DNA damage" is in contradiction to the general 3' end processing defect following DNA-damage that the authors cite. This statement is only understandable with the prior knowledge of p53 mRNA being able to escape this inhibition. I feel it would be helpful to rephrase this.

    Figure 1 E): shifting the entire axis up, is to my reading counterintuitive. I would show all graphs at the same scale.

    Williamson et al (Svejstrup) 2017 showed through DRB-washed PRO-seq profiles that Pol II elongation speed is reduced for at least 8 hrs following UV-exposure, resulting in depletion from Pol II in gene bodies and concentration towards the gene beginnings (transcript start sites, TSS). It would be helpful to have an idea how the transcriptional profile looks on p53 at the time point of analysis - and at which time point after DNA-damage insult the 3' end processing inhibition starts. Such study could also form the beginning of a more in-depth analysis on how the CoTC is mediated. Such an in-depth analysis should also probe the chromatin crosslinking of RNA Pol II, as well as 3' end factors at the regular PAS and downstream Co-TC element. The fact that the smFISH shows signal for the downstream probes of Rad53, suggests that Pol II regularly transcribes to these positions. Could there be another PAS-dependent termination signal in further downstrea areas?

    Figure 5) the sequencing procedure should be explained better in the main text. From the text it is not clear, what sort of sequencing was performed.

    Referees cross-commenting

    @ Reviewer #1:

    Generally agree with Reviewer 1.

    1. Figure 3B : I agree that this experiment would benefit from better description. In fact, wouldnt one expect to be there signal in Figure 3D after UV, since cleavage is inhibited? Unless Williamson et al is taken into account, showing that transcription is generally slowed.

    @ Reviewer #3:

    Generally find all these suggestions are very valid and would increase the value of the manuscript. I am not sure if I understand correctly/agree with two comments:

    If understood correctly, this reviewer suggests that there is no Pcf11 under UV treatment conditions as suggested in Figure 1A. However, Figure 1B might show a longer Western Blot exposure or have more material loaded, showing that there is some Pcf11 available for si-mediated knockdown.

    Figure 5 Panel B. I would agree that this panel is not very well explained. My interpretation so far has been in quartiles (left top, less cleaved, less total; top right less cleaved, more total upon UV versus bottom left more cleaved, downregulated and bottom right more cleaved upreagulated upon UV). In which case a significant number of transcripts is no cleaved upon UV. To help with interpretation at least a longer legend could be added.

    Significance

    This manuscript is overall convincing and adds more gravitas to the highly debated observation of co-transcriptional cleavage events. Although this study is by no means mechanistically exhaustive, it shows that the proposed mechanism may be true for the genes of DNA-damage repair factors that need to be "exempted" from the general DNA-damage-induced inhibition of 3' end cleavage. This opens up the exciting possibility that co-transcriptional elements can be used under specific, controlled environmental conditions. Although alternative explanations are possible to explain these pre-mRNA's release from the chromatin, 3' end processing at the regular poly(A) signal is for these RNAs clearly inhibited.

    For a complete classification as Co-TC element however, additional experiments would be required. I am not adding these to the major or minor points, as these in my eyes would constitute a new story. The original literature on CoTC (West et al 2004, Teixeira et al 2004), posed that a Co-TC event provides a 5'phospho entry site that could be used by a molecular torpedo (Xrn2). Part of the controversy about Co-TC cleavage is the question of how such a 5'phospho-end could chemically be generated by autocatalytic cleavage. To substantiate the claim that these elements are indeed Co-TC cleavage events either generated by auto-catalytic cleavage or another enzymatic function, the authors should test if they promote termination in this homologous, as well as a heterologous context.

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    Referee #1

    Evidence, reproducibility and clarity

    Sfaxi et al., significantly extended a current knowledge of the mechanism and the function of co-transcriptional RNA cleavage (CoTC). First, the authors focused on TP53 genes to study the CoTC. They showed TP53 transcripts are cleaved independently of PCF11 and Pol II CTD Ser2 phosphorylation in the UV-treated cells, concluding RNA cleavage at polyadenylation site (PAS) of the TP53 gene occurs post-transcriptionally. This strongly suggests that TP53 gene transcription termination is regulated by CoTC. They also showed a biological importance of the CoTC on TP53 gene expression. Depletion of the potential TP53 CoTC genomic region impaired mRNA and protein levels of p53 and its target p21. This deregulated G1-S phase progression in cell cycle following UV treatment. Finally they extended these findings to other genes by the novel screening approach of the CoTC.

    Minor comments:

    1. P4, Second paragraph, Another model~; Two more papers need to be cited. "Dye and Proudfoot 2001 Cell" "West et al., 2008 Mol Cell"
    2. Figure 3B; The authors should explain more about foci detected by probes A and B.
    3. P10, Last line, strong decrease~; (Figures 4E and ~) -> (Figure 4E, right panels, and~)
    4. Figure 5C; The author should show the entire image of the RNA-seq reads in the gene region, but not just in the windows described in Figure 5A. Also, TP53 and GAPDH genes need to be shown for the controls.

    Referees cross-commenting

    I totally agree with Reviewer #2.

    Significance

    Overall this paper is well described and written. In my view, this will bring important information to the transcription termination field.

    Note, I am not sure that the authors need to include the generality of the CoTC in Figures 5 and 6 since their RNA-seq analysis and its validation for biological functions are incomplete. Therefore I feel that focusing on the TP53 gene may enhance the impact of this paper.

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