S. pombe Rtf2 is important for replication fork barrier activity of RTS1 via splicing of Rtf1

Abstract

Arrested replication forks, when restarted by homologous recombination, result in error-prone DNA syntheses and non-allelic homologous recombination. Fission yeast RTS1 is a model fork barrier used to probe mechanisms of recombination-dependent restart. RTS1 barrier activity is entirely dependent on the DNA binding protein Rtf1 and partially dependent on a second protein, Rtf2. Human RTF2 was recently implicated in fork restart, leading us to examine fission yeast Rtf2's role in more detail. In agreement with previous studies, we observe reduced barrier activity upon rtf2 deletion. However, we identified Rtf2 to be physically associated with mRNA processing and splicing factors and rtf2 deletion to cause increased intron retention. One of the most affected introns resided in the rtf1 transcript. Using an intronless rtf1 we observed no reduction in RFB activity in the absence of Rtf2. Thus, Rtf2 is essential for correct rtf1 splicing to allow optimal RTS1 barrier activity.

Data availability

Sequence data is available under GEO dataset GSE192344.

The following data sets were generated

Article and author information

Author details

  1. Alice M Budden

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Murat Eravci

    Department of Biochemistry and Biomedicine, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Adam T Watson

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Eduard Campillo-Funollet

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Antony W Oliver

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2912-8273
  6. Karel Naiman

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    For correspondence
    karel.naiman@inserm.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3594-1516
  7. Antony M Carr

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    For correspondence
    A.M.Carr@sussex.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2028-2389

Funding

Wellcome Trust (11047/Z/15/Z)

  • Antony M Carr

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2023, Budden et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 377
    views
  • 55
    downloads
  • 2
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Alice M Budden
  2. Murat Eravci
  3. Adam T Watson
  4. Eduard Campillo-Funollet
  5. Antony W Oliver
  6. Karel Naiman
  7. Antony M Carr
(2023)
S. pombe Rtf2 is important for replication fork barrier activity of RTS1 via splicing of Rtf1
eLife 12:e78554.
https://doi.org/10.7554/eLife.78554

Share this article

https://doi.org/10.7554/eLife.78554

Further reading

    1. Cell Biology
    2. Developmental Biology
    Sofía Suárez Freire, Sebastián Perez-Pandolfo ... Mariana Melani
    Research Article

    Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.

    1. Cell Biology
    2. Developmental Biology
    Heungjin Ryu, Kibum Nam ... Jung-Hoon Park
    Research Article

    In most murine species, spermatozoa exhibit a falciform apical hook at the head end. The function of the sperm hook is not yet clearly understood. In this study, we investigate the role of the sperm hook in the migration of spermatozoa through the female reproductive tract in Mus musculus (C57BL/6), using a deep tissue imaging custom-built two-photon microscope. Through live reproductive tract imaging, we found evidence indicating that the sperm hook aids in the attachment of spermatozoa to the epithelium and facilitates interactions between spermatozoa and the epithelium during migration in the uterus and oviduct. We also observed synchronized sperm beating, which resulted from the spontaneous unidirectional rearrangement of spermatozoa in the uterus. Based on live imaging of spermatozoa-epithelium interaction dynamics, we propose that the sperm hook plays a crucial role in successful migration through the female reproductive tract by providing anchor-like mechanical support and facilitating interactions between spermatozoa and the female reproductive tract in the house mouse.