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  1. Evaluation Summary:

    DNA replication forks can become arrested either in a cellular program or by accident. In fission yeast, DNA replication fork arrests at the locus called the RTS1 are mediated by a DNA binding protein, Rtf1. In this paper, by combining genetics, proteomics, and genomics approaches, the authors nicely showed the role of Rtf2 as a fork barrier to mediate the splicing of rtf1 mRNA. The splicing-mediated control of protein abundance provides a new regulatory mechanism for the programmed DNA replication barrier.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    This paper describes the characterization of fission yeast Rtf2 protein, which was previously shown to work on stalling of DNA replication forks at a fork barrier sequence, RTS1 in the yeast. By using a novel technique called Polymerase usage sequencing (Pu-seq), the authors confirmed the role of Rtf2 in the fork stalling at the RTS1 site (by looking at a change of DNA polymerase usage at the site). To elucidate the role of Rtf2 in the DNA replication fork stalling, the authors identified proteins that interact with Rtf2 protein and identified proteins involved in mRNA splicing. Indeed, cDNA sequencing revealed a defect in intron removal in some mRNAs in the rtf2 mutant. Interestingly, among RNAs affected in the absence of Rtf2, the authors found the Rtf1 RNA as a target for Rtf2 splicing. Indeed, the removal of the second intron of Rtf2 RNA is defective in the rtf2 mutant. Finally, the authors showed that an intronless Rtf1 gene suppressed the defect in the rtf2 mutant in the fork stalling.

    The experiments in the paper have been carried out in good quality and most of the data are convincing. The paper revealed the unique role of Rtf2 in the splicing of Rtf1 RNA.

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  3. Reviewer #2 (Public Review):

    Human RTF2 is a protein whose degradation from stalled forks is necessary to promote replication restart and maintain genome stability. The S. pombe homologue Rtf2 has been previously described as a factor that binds to the replication fork barrier (RFB) RTS1 to reinforce its barrier activity and limit replication restart. In this work, the authors have conducted a careful analysis of the function of Rtf2 in regulating the activity of the RFB. First, the authors have confirmed that the deletion of rtf2 reduces the RFB activity and leads to a less frequent replication restart using genetics and genomic assays. Then, they have established that Rtf2 does not regulate the activity of the RFB by direct binding to part A of the RTS1 sequence. To understand further the mechanism by which Rtf2 regulates the activity of the RFB, proteomics approaches were performed to reveal that most of the Rtf2 partners are related to mRNA processing and splicing. Then, the authors have identified that the absence of Rtf2 leads to the accumulation of mis-spliced rtf1 mRNA that encodes the Rtf1 protein that binds the RTS1 sequence to ensure replication fork arrest at the RFB. In a final demonstration, it is shown that when an intron-free Rtf1 form is expressed, Rtf2 is no longer necessary to regulate the activity of the RFB. Overall, the authors have conducted a remarkable work to disentangle the function of Rtf2 in regulating the activity of the RFB and establish that Rtf2 is a factor necessary for the correct splicing of Rtf1, thus regulating indirectly the activity of the RFB.

    This is a well-conducted study with robust conclusions based on solid data. The article is well written and the rationale of the work is logical and easy to follow. There is no major weakness.

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  4. Reviewer #3 (Public Review):

    Fission yeast RTS1 barrier activity is partially dependent on Rtf2 protein and human RTF2 was recently implicated in impeding fork restart, however, the exact role of Rtf2 at the stalled fork in fission yeast is not well understood. In this manuscript, the authors used a sophisticated system to control fork stalling, combined with the high-resolution Pu-seq to probe fork dynamics, they examined the detailed function of Rtf2 in fission yeast and confirmed that Rtf2 deletion reduces replication fork restart at RTS1. To further dissect the mechanism of Rtf2's function, surprisingly, they identified Rtf2 to be physically associated with mRNA processing and splicing factors and rtf2 deletion to cause increased intron retention. Indeed, they demonstrated that the presence of Rtf2 is essential for the correct splicing of Rtf1 to allow efficient barrier activity at RTS1, which is complemented by an intronless Rtf1 in the absence of Rtf2.

    The conclusions of this paper are mostly well supported by data, but additional discussions and some clarification of data analysis are needed.

    1. The authors identified Rtf2 as physically associating with general splicing factors, thus having a broader impact on splicing overall as shown in Fig 3A & 3B. Rtf1 is one of the many targets affected by intron retention, which was further characterized by reduced GC richness at the branch point and 3' SS as in Fig S3C. Please clarify the GC richness on Rtf1 introns affected for clarity. It is understandable that in a designed fork barrier model, the impact of Rtf1 stands out. It remains to be clarified if other mis-spliced genes may function in other biological processes, maybe explaining why they need to be finetuned by Rtf2, a short discussion on this will suffice, as well as some discussion about the functional divergence between yeast, plant and human Rtf2 homologs.

    2. The authors used the Pu-seq method in the RTS1 system to track non-canonical RDR forks. One caveat is that RTS1 is an artificially constructed system, whether impeded replication forks work in the same pattern in the whole genome needs to be discussed.

    3. A ChIP assay for Rtf1 at RTS1 (region B) between rtf2+ and rtf2∆ will help further prove the claimed impact of intron retention of Rtf1 at RTS1 barrier activity. If ChIP is not possible due to a lack of appropriate antibodies, it is ok to speculate but still needs to be made clear in the text.

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