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

Publication 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

  • 1,907
    Page views
  • 306
    Downloads
  • 12
    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
    2. Structural Biology and Molecular Biophysics

    Molecular basis for the role of disulfide-linked αCTs in the activation of insulin-like growth factor 1 receptor and insulin receptor

    Jie Li, Jiayi Wu ... Eunhee Choi
    Research Article Updated

    The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the Γ-shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.

    1. Cell Biology
    2. Chromosomes and Gene Expression

    Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

    Zhanwu Zhu, Jingjing Liu ... Bo Cheng
    Research Article

    Dynamic regulation of transcription is crucial for the cellular responses to various environmental or developmental cues. Gdown1 is a ubiquitously expressed, RNA polymerase II (Pol II) interacting protein, essential for the embryonic development of metazoan. It tightly binds Pol II in vitro and competitively blocks the binding of TFIIF and possibly other transcriptional regulatory factors, yet its cellular functions and regulatory circuits remain unclear. Here, we show that human GDOWN1 strictly localizes in the cytoplasm of various types of somatic cells and exhibits a potent resistance to the imposed driving force for its nuclear localization. Combined with the genetic and microscope-based approaches, two types of the functionally coupled and evolutionally conserved localization regulatory motifs are identified, including the CRM1-dependent nucleus export signal (NES) and a novel Cytoplasmic Anchoring Signal (CAS) that mediates its retention outside of the nuclear pore complexes (NPC). Mutagenesis of CAS alleviates GDOWN1’s cytoplasmic retention, thus unlocks its nucleocytoplasmic shuttling properties, and the increased nuclear import and accumulation of GDOWN1 results in a drastic reduction of both Pol II and its associated global transcription levels. Importantly, the nuclear translocation of GDOWN1 occurs in response to the oxidative stresses, and the ablation of GDOWN1 significantly weakens the cellular tolerance. Collectively, our work uncovers the molecular basis of GDOWN1’s subcellular localization and a novel cellular strategy of modulating global transcription and stress-adaptation via controlling the nuclear translocation of GDOWN1.

    1. Cell Biology
    2. Neuroscience

    The NAD+ precursor NMN activates dSarm to trigger axon degeneration in Drosophila

    Arnau Llobet Rosell, Maria Paglione ... Lukas Jakob Neukomm
    Research Article

    Axon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD+. SARM1 NADase activity is activated by the NAD+ precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury. However, it remains unclear whether NMN is also a key mediator of axon degeneration and dSarm activation in flies. Here, we demonstrate that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD+ metabolic flux by lowering NMN, while NAD+ remains unchanged in vivo. Increased NMN synthesis, by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT), leads to faster axon degeneration after injury. We also show that NMN-induced activation of dSarm mediates axon degeneration in vivo. Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD+-synthesizing enzyme dNmnat. Our results reveal a critical role for NMN in neurodegeneration in the fly, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar to the interference with other essential mediators of axon degeneration in Drosophila.