Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation
- University of Rome, Italy
- University of Exeter, United Kingdom
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
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.
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.
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Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation
eLife 7:e40325.
https://doi.org/10.7554/eLife.40325
Further reading
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- Cell Biology
- Chromosomes and Gene Expression
Meiotic crossover recombination is essential for both accurate chromosome segregation and the generation of new haplotypes for natural selection to act upon. This requirement is known as crossover assurance and is one example of crossover control. While the conserved role of the ATPase, PCH-2, during meiotic prophase has been enigmatic, a universal phenotype when pch-2 or its orthologs are mutated is a change in the number and distribution of meiotic crossovers. Here, we show that PCH-2 controls the number and distribution of crossovers by antagonizing their formation. This antagonism produces different effects at different stages of meiotic prophase: early in meiotic prophase, PCH-2 prevents double-strand breaks from becoming crossover-eligible intermediates, limiting crossover formation at sites of initial double-strand break formation and homolog interactions. Later in meiotic prophase, PCH-2 winnows the number of crossover-eligible intermediates, contributing to the designation of crossovers and ultimately, crossover assurance. We also demonstrate that PCH-2 accomplishes this regulation through the meiotic HORMAD, HIM-3. Our data strongly support a model in which PCH-2’s conserved role is to remodel meiotic HORMADs throughout meiotic prophase to destabilize crossover-eligible precursors and coordinate meiotic recombination with synapsis, ensuring the progressive implementation of meiotic recombination and explaining its function in the pachytene checkpoint and crossover control.
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- Cell Biology
In animals, mitosis involves the breakdown of the nucleus. The reassembly of a nucleus after mitosis requires the reformation of the nuclear envelope around a single mass of chromosomes. This process requires Ankle2 (also known as LEM4 in humans) which interacts with PP2A and promotes the function of the Barrier-to-Autointegration Factor (BAF). Upon dephosphorylation, BAF dimers cross-bridge chromosomes and bind lamins and transmembrane proteins of the reassembling nuclear envelope. How Ankle2 functions in mitosis is incompletely understood. Using a combination of approaches in Drosophila, along with structural modeling, we provide several lines of evidence that suggest that Ankle2 is a regulatory subunit of PP2A, explaining how it promotes BAF dephosphorylation. In addition, we discovered that Ankle2 interacts with the endoplasmic reticulum protein Vap33, which is required for Ankle2 localization at the reassembling nuclear envelope during telophase. We identified the interaction sites of PP2A and Vap33 on Ankle2. Through genetic rescue experiments, we show that the Ankle2/PP2A interaction is essential for the function of Ankle2 in nuclear reassembly and that the Ankle2/Vap33 interaction also promotes this process. Our study sheds light on the molecular mechanisms of post-mitotic nuclear reassembly and suggests that the endoplasmic reticulum is not merely a source of membranes in the process, but also provides localized enzymatic activity.
