1. Cell Biology

Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

  1. Claudia Pellacani
  2. Elisabetta Bucciarelli
  3. Fioranna Renda
  4. Daniel Hayward
  5. Antonella Palena
  6. Jack Chen
  7. Silvia Bonaccorsi
  8. James G Wakefield
  9. Maurizio Gatti  Is a corresponding author
  10. Maria Patrizia Somma  Is a corresponding author
  1. University of Rome, Italy
  2. University of Exeter, United Kingdom
https://doi.org/10.7554/eLife.40325
Share this article
Cite this article
  1. Claudia Pellacani
  2. Elisabetta Bucciarelli
  3. Fioranna Renda
  4. Daniel Hayward
  5. Antonella Palena
  6. Jack Chen
  7. Silvia Bonaccorsi
  8. James G Wakefield
  9. Maurizio Gatti
  10. Maria Patrizia Somma
(2018)
Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation
eLife 7:e40325.
https://doi.org/10.7554/eLife.40325
  2. Download BibTeX
  3. Download .RIS

Abstract

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 minute, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.

Data availability

All data generated or analyzed during this study are included in the manuscript and Supplementary File 1. The full dataset for proteomic analyses reported in Figures 6 and Supplementary File 1 can be found at https://www.thewakefieldlab.com/ms; the significantly reduced protein IDs for each RNAi experiment were interrogated using a Gene Ontology (GO) classifier (GOTermMapper (https://go.princeton.edu/cgi-bin/GOTermMapper), concentrating on the GO terms ""cell division"" (GO:0051301), ""mitotic cell cycle"" (GO:0000278) and ""chromosome segregation"" (GO:0007059). Source data files have been provided for Figures 1, 4, 5, 7, 8, and 9.

Article and author information

Author details

  1. Claudia Pellacani

    Department of Biology and Biotechnology Charles Darwin, University of Rome, Rome, Italy
    Competing interests
    The authors declare that no competing interests exist.

  2. Elisabetta Bucciarelli

    Institute of Molecular Biology and Pathology, University of Rome, Rome, Italy
    Competing interests
    The authors declare that no competing interests exist.

  3. Fioranna Renda

    Department of Biology and Biotechnology Charles Darwin, University of Rome, Rome, Italy
    Competing interests
    The authors declare that no competing interests exist.

  4. Daniel Hayward

    Biosciences/Living Systems Institute, University of Exeter, Exeter, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Antonella Palena

    Institute of Molecular Biology and Pathology, University of Rome, Rome, Italy
    Competing interests
    The authors declare that no competing interests exist.

  6. Jack Chen

    Biosciences/Living Systems Institute, University of Exeter, Exeter, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  7. Silvia Bonaccorsi

    Department of Biology and Biotechnology Charles Darwin, University of Rome, Roma, Italy
    Competing interests
    The authors declare that no competing interests exist.

  8. James G Wakefield

    Biosciences/Living Systems Institute, University of Exeter, Exeter, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  9. Maurizio Gatti

    Department of Biology and Biotechnology Charles Darwin, University of Rome, Rome, Italy
    For correspondence
    maurizio.gatti@uniroma1.it
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3777-300X

  10. Maria Patrizia Somma

    Institute of Molecular Biology and Pathology, University of Rome, Rome, Italy
    For correspondence
    patrizia.somma@uniroma1.it
    Competing interests
    The authors declare that no competing interests exist.

Funding

Associazione Italiana per la Ricerca sul Cancro (IG16020)

  • Maurizio Gatti

Ministero dell'Istruzione, dell'Università e della Ricerca

  • Silvia Bonaccorsi

Biotechnology and Biological Sciences Research Council (BB/K017837/1)

  • James G Wakefield

Associazione Italiana per la Ricerca sul Cancro (IG20528)

  • Maurizio Gatti

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

Reviewing Editor

  1. Jon Pines, Institute of Cancer Research Research, United Kingdom

Version history

  1. Received: July 22, 2018
  2. Accepted: November 14, 2018
  3. Accepted Manuscript published: November 26, 2018 (version 1)
  4. Version of Record published: December 10, 2018 (version 2)

Copyright

© 2018, Pellacani 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

  • 2,057
    Page views
  • 321
    Downloads
  • 14
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

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. Claudia Pellacani
  2. Elisabetta Bucciarelli
  3. Fioranna Renda
  4. Daniel Hayward
  5. Antonella Palena
  6. Jack Chen
  7. Silvia Bonaccorsi
  8. James G Wakefield
  9. Maurizio Gatti
  10. Maria Patrizia Somma
(2018)
Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation
eLife 7:e40325.
https://doi.org/10.7554/eLife.40325

Categories and tags

Research organism

Further reading

    1. Cell Biology

    PM2.5 leads to adverse pregnancy outcomes by inducing trophoblast oxidative stress and mitochondrial apoptosis via KLF9/CYP1A1 transcriptional axis

    Shuxian Li, Lingbing Li ... Xietong Wang
    Research Article

    Epidemiological studies have demonstrated that fine particulate matter (PM2.5) is associated with adverse obstetric and postnatal metabolic health outcomes, but the mechanism remains unclear. This study aimed to investigate the toxicological pathways by which PM2.5 damaged placental trophoblasts in vivo and in vitro. We confirmed that PM2.5 induced adverse gestational outcomes such as increased fetal mortality rates, decreased fetal number and weight, damaged placental structure, and increased apoptosis of trophoblasts. Additionally, PM2.5 induced dysfunction of the trophoblast cell line HTR8/SVneo, including in its proliferation, apoptosis, invasion, migration and angiogenesis. Moreover, we comprehensively analyzed the transcriptional landscape of HTR8/SVneo cells exposed to PM2.5 through RNA-Seq and observed that PM2.5 triggered overexpression of pathways involved in oxidative stress and mitochondrial apoptosis to damage HTR8/SVneo cell biological functions through CYP1A1. Mechanistically, PM2.5 stimulated KLF9, a transcription factor identified as binding to CYP1A1 promoter region, which further modulated the CYP1A1-driven downstream phenotypes. Together, this study demonstrated that the KLF9/CYP1A1 axis played a crucial role in the toxic progression of PM2.5 induced adverse pregnancy outcomes, suggesting adverse effects of environmental pollution on pregnant females and putative targeted therapeutic strategies.

    1. Cell Biology
    2. Microbiology and Infectious Disease

    PerTurboID, A targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria causing parasites

    Heledd Davies, Hugo Belda ... Moritz Treeck
    Tools and Resources

    Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

    1. Cell Biology

    CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis

    Bo Wang, Zheyong Liang ... Peijun Liu
    Research Article

    The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.