Human Cactin Interacts with DHX8 and SRRM2 to Assure Efficient Pre-Mrna Splicing and Sister Chromatid Cohesion Isabella M
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© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068 RESEARCH ARTICLE Human cactin interacts with DHX8 and SRRM2 to assure efficient pre-mRNA splicing and sister chromatid cohesion Isabella M. Y. Zanini1,*, Charlotte Soneson2,‡, Luca E. Lorenzi1 and Claus M. Azzalin1,§ ABSTRACT development (Atzei et al., 2010a). To date, cactin orthologs have Cactins constitute a family of eukaryotic proteins broadly conserved been studied also in Toxoplasma gondii, Litopenaeus vannamei, from yeast to human and required for fundamental processes such Arabidopsis thaliana, Caenorhabditis elegans, and Danio rerio as cell proliferation, genome stability maintenance, organismal (Atzei et al., 2010b; Baldwin et al., 2013; Cecchetelli et al., 2016; development and immune response. Cactin proteins have been Doherty et al., 2014; LaBonty et al., 2014; Szatanek et al., 2012; found to associate with the spliceosome in several model organisms, Tannoury et al., 2010; Zhang et al., 2014). In these organisms, nevertheless their molecular functions await elucidation. Here we cactin loss-of-function is associated with compromised cell viability show that depletion of human cactin leads to premature sister and proliferation, and with developmental defects, highlighting the chromatid separation, genome instability and cell proliferation arrest. essentiality of cactin proteins. In Schizosaccharomyces pombe, Moreover, cactin is essential for efficient splicing of thousands of ablation of Cactin in fission yeast 1 (Cay1) only mildly affects Δ pre-mRNAs, and incomplete splicing of the pre-mRNA of sororin proliferation in standard culture conditions. However, cay1 cells (also known as CDCA5), a cohesin-associated factor, is largely accumulate aberrant chromosome structures and stop dividing when responsible for the aberrant chromatid separation in cactin-depleted shifted to cold temperatures (Lorenzi et al., 2015). cells. Lastly, cactin physically and functionally interacts with the Although the molecular functions of cactins remain elusive, spliceosome-associated factors DHX8 and SRRM2. We propose protein interaction and localization studies have suggested that cellular complexes comprising cactin, DHX8 and SRRM2 connections with pre-mRNA splicing. Mass-spectrometry-based sustain precise chromosome segregation, genome stability and cell analysis of purified human and Drosophila spliceosomes identified proliferation by allowing faithful splicing of specific pre-mRNAs. Our Cactin as a component of the catalytically active spliceosome data point to novel pathways of gene expression regulation complex C (Bessonov et al., 2008; Herold et al., 2009; Jurica et al., dependent on cactin, and provide an explanation for the pleiotropic 2002; Rappsilber et al., 2002; Zhou et al., 2002). Moreover, analysis dysfunctions deriving from cactin inactivation in distant eukaryotes. of the protein interactome of the C. elegans cactin ortholog, CACN- 1, revealed interactions with several spliceosome components KEY WORDS: Cactin, Pre-mRNA splicing, Sororin, Sister chromatid (Doherty et al., 2014). Similarly, Drosophila Cactin interacts with cohesion, DHX8, SRRM2 the core spliceosome factor SmB protein (Giot et al., 2003) and the cactin interactor IκBL forms complexes with spliceosomal proteins INTRODUCTION (An et al., 2013). In A. thaliana, cactin colocalizes with the two The cactin protein family comprises evolutionarily conserved splicing factors RSP31 and SR45 within nuclear speckles (Baldwin polypeptides involved in seemingly disparate cellular processes. et al., 2013) and RNPS1, the human ortholog of SR45, also interacts Isolated at first as an antigen recognized by autologous antibodies in with cactin (Ewing et al., 2007). Functional studies in S. pombe further sera from human patients with renal-cell carcinoma (Scanlan et al., support a link with pre-mRNA splicing. cay1Δ cells inefficiently 1999), cactin was subsequently identified in and named after a two- splice the pre-mRNA of the telomeric factor Rap1, which promotes hybrid screening performed using the Drosophila melanogaster heterochromatin establishment at telomeres and restricts telomerase- I-κB protein Cactus as bait (Lin et al., 2000). Overexpression of mediated telomere elongation (Lorenzi et al., 2015; Miller et al., Cactin in a Cactus-compromised background enhanced cactus 2005). As a consequence, in cay1Δ cells, Rap1 protein levels are phenotypes including embryonic lethality and embryo severely diminished, heterochromatin-mediated telomere silencing is ventralization (Lin et al., 2000). Human cactin was later found to weakened and telomeres are excessively elongated by telomerase physically and functionally interact with IκB-like protein (IκBL) (Lorenzi et al., 2015). cay1Δ cells also accumulate unprocessed and to be part of a negative feedback loop that controls NFκB precursor transcripts from Tf2 retrotransposons, a feature shared with transcriptional response (Suzuki et al., 2016), suggesting a independent yeast splicing mutants (Lorenzi et al., 2015). conserved function for cactins in immune response and Here, we show that depletion of human cactin in various cultured immortal cell types leads to premature sister chromatid separation, 1Institute of Biochemistry (IBC), Department of Biology, Eidgenössische Technische genome instability and cell proliferation arrest. Deep sequencing of Hochschule Zürich (ETHZ), Zürich CH-8093, Switzerland. 2Bioinformatics Core Facility, SIB Swiss Institute of Bioinformatics, Lausanne CH-1015, Switzerland. the transcriptome of cactin-depleted cells reveals that thousands of *Present address: Department of Molecular Mechanisms of Disease, University of pre-mRNAs are incompletely spliced. In particular, we find that the Zürich, Zürich CH-8057, Switzerland. ‡Present address: Institute for Molecular Life pre-mRNA of sororin (also known as CDCA5), a cohesin- Sciences, University of Zürich and SIB Swiss Institute of Bioinformatics, Zürich CH-8057, Switzerland. associated factor, is aberrantly spliced and sororin protein levels are diminished upon cactin depletion. Expression of a sororin §Author for correspondence ([email protected]) cDNA in cactin-depleted cells is sufficient to largely restore normal C.S., 0000-0003-3833-2169; C.M.A., 0000-0002-9396-1980 chromatid cohesion, revealing a fundamental role for sororin dysfunction in the cellular defects associated with cactin Received 17 June 2016; Accepted 26 December 2016 deficiency. Lastly, we show that cactin physically and Journal of Cell Science 767 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068 functionally interacts with the spliceosome-associated factors (Fig. 1A, Fig. 2A), when cell proliferation was already evidently DHX8 and SRRM2. Our data indicate that cactin supports normal impaired. Fluorescence-activated cell sorting (FACS) of propidium- cellular physiology by promoting efficient splicing of a multitude of iodide-stained cells confirmed a proliferation defect in cactin- pre-mRNAs, and further support the emerging idea that cactin depleted cells, which ultimately accumulated in S and G2/M phases proteins are functional components of the splicing machineries of in HeLa cells (Fig. 1B) and in G2/M phase in U2OS cells (Fig. 2B). distant eukaryotes. As shown by indirect immunofluorescence, cactin-depleted cells displayed increased frequencies of nuclear foci containing the DNA RESULTS damage marker 53BP1 (Fig. 1C, Fig. 2C). Similarly, another DNA Cactin supports cell proliferation, genome stability, nuclear damage marker, histone H2AX phosphorylated at serine 139 morphology and sister chromatid cohesion (γH2AX), accumulated upon cactin depletion, mostly in cells in late To unveil the functions exerted by cactin we transfected cervical S and G2/M phases, as shown by flow cytometric analysis carcinoma HeLa and osteosarcoma U2OS cells with two combining anti-γH2AX antibodies and 5-ethynyl-2′-deoxyuridine independent siRNAs directed against the 3′-UTR of cactin (EdU) incorporation (Fig. 1D, Fig. 2D). We then performed pulse- mRNA (siCacD and siCacE) and harvested cells 24, 48 and 72 h field gel electrophoresis (PFGE) of undigested genomic DNA and later. Approximately 70% and 80% protein depletion was achieved uncovered broken DNA in cactin-depleted cells (Fig. S1A), in HeLa and U2OS cells, respectively, at the latest time points implying that 53BP1 foci and γH2AX accrue at least in part in Fig. 1. Cactin is required for cell cycle progression, genome stability and normal nuclear morphology in HeLa cells. HeLa cells were transfected with siCacD, siCacE and control (siCtrl) siRNAs and harvested 24, 48 and 72 h post-transfections. (A) Western blot analysis of cactin depletion in HeLa cells transfected with the indicated siRNAs and. Actin serves as loading control. siCtrl, siRNA control. Percentage values beneath blots indicate the amount of cactin remaining in the depleted samples expressed as fraction of siCtrl-transfected samples after normalization to the actin signal. (B) FACS analysis of propidium- iodide-stained cells. The bar graph at the top shows the fractions of cells in the different phases of the cell cycle. (C) Examples of indirect immunofluorescence analysis of 53BP1 foci. DAPI-stained DNA is shown in gray, 53BP1 in green. The graph on the right shows the percentages of 53BP1-positive cells (53BP1+, cells with at least five foci). (D) Three-channel flow-cytometry-based analysis of DNA content (x axis),