© 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 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 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 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 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), 5-ethynyl-2′-deoxyuridine incorporation (EdU; y axis) and γH2AX levels (in red). The percentages of cells positive for γH2AX are indicated. (E) Example of siCacD-transfected cell with an abnormal nucleus observed after DAPI staining (top panel) and under phase contrast (bottom panel). The graph on the right shows the percentages of cells with abnormal nuclei. Data in C,E represented as mean±s.e.m. from three independent experiments. At least 100 nuclei were scored for each sample in each experiment. *P<0.05, **P<0.01

(relative to siCtrl at 24 h; two-tailed Student’s t-test). Journal of Cell Science

768 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068

Fig. 2. Cactin is required for cell cycle progression, genome stability and normal nuclear morphology in U2OS cells. U2OS 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 U2OS cells transfected with the indicated siRNAs and harvested 24, 48 and 72 h post transfection. 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 fraction 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 at the bottom shows the percentages of 53BP1 positive cells (53BP1+, cells with at least five foci). (D) Flow-cytometry-based analysis of DNA content (x axis), EdU incorporation (y axis) and γH2AX levels (in red). The percentages of cells positive for γH2AX are indicated. The three panels on top show control profiles of cells treated with bleomycin, camptothecin (CPT) or left untreated (NT). (E) Example of siCacD-transfected cell with an abnormal nucleus observed after DAPI staining (left panel) and under phase contrast (right panel). The graph at the bottom shows the percentages of cells with abnormal nuclei. Data in C,E represented as mean±s.e.m. from three independent experiments. At least 100 nuclei were scored for each sample in each experiment. *P<0.05, **P<0.01, ***P<0.001 (relative to siCtrl at 24 h; two-tailed Student’s t-test). response to DNA double-stranded breaks. Because Cay1 deficiency cactin-depleted cells: (1) 53BP1 foci did not preferentially colocalize in fission yeast is associated with aberrant telomere maintenance with telomeres (Fig. S1B); (2) the levels of several telomeric factors, (Lorenzi et al., 2015), the DNA damage observed in cactin-depleted including human RAP1 (officially known as TERF2IP), were not

cells could be elicited by dysfunctional telomeres. However, in noticeably altered (Fig. S1C); and (3) telomere length and silencing Journal of Cell Science

769 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068 were normal (data not shown). Thus, contrarily to what was observed retention in the cactin-depleted samples. Affected participate in fission yeast, cactin deficiency seems not to majorly disturb in different cellular processes, with a striking enrichment in nucleic telomere maintenance in human cells. acid synthesis and regulation, and in ubiquitin ligase activity The fact that nucleotide incorporation defects preceded process (Fig. S3B). This shows that cactin supports pre-mRNA accumulation of γH2AX (Fig. 1D, Fig. 2D), together with the splicing of a multitude of pre-mRNAs and suggests that cactin appearance of DNA breaks (Fig. S1A) suggested that cell cycle supports normal cellular physiology by regulating various cellular arrest in cactin-depleted cells could be triggered by activation of pathways. checkpoints responding to DNA damage caused by impaired DNA replication. Indeed, KAP1 phosphorylation at serine 824 and CHK1 Cactin promotes splicing of sororin pre-mRNA phosphorylation at serine 345, which are markers for activation of While searching for mis-spliced transcripts that could account for the DDR checkpoint kinases ATM and ATR, respectively (Liu et al., the cellular defects arising upon cactin depletion, the cohesin 2000; Ziv et al., 2006), mildly accumulated in cactin-depleted cells accessory factor sororin (CDCA5 in Table S1) caught our attention (Fig. S1D). Similarly, phosphorylation of the single-stranded DNA- because erroneous splicing of its pre-mRNA was previously binding protein RPA32 at serine 33, a sensitive marker for DNA reported to cause premature separation of sister chromatids (Oka replication defects and S-phase checkpoint activation (Olson et al., et al., 2014; Sundaramoorthy et al., 2014; van der Lelij et al., 2014; 2006), was detected, although at very low levels, in cactin-depleted Watrin et al., 2014), a major phenotype induced by cactin depletion. cells (Fig. S1D,E). We thus conclude that cactin depletion induces In our RNA-seq data, reads corresponding to sororin intron 1 were mild replication stress and activation of DNA damage checkpoints, over-represented in cactin-depleted samples as compared with which are likely to contribute to cell cycle progression impairment. control samples, as well as with sororin introns 2–5 both in cactin- Nevertheless, given the very low levels of phosphorylation of depleted and control samples (Fig. 4B,C). Consistently, in northern KAP1, CHK1 and RPA32, we do not exclude that other blot analysis of total RNA from depleted cells, full-length, mechanisms not relying on DNA damage checkpoint activation unprocessed sororin pre-mRNA only mildly accumulated, could contribute to the observed proliferation defects. whereas the majority of sororin transcripts were slightly longer We then tested whether cactin is required for normal chromosome than in control samples (Fig. 4D). Reverse-transcription (RT)-PCR segregation, as is the case in fission yeast cells grown in the cold analysis confirmed robust retention of intron 1 upon depletion of (Lorenzi et al., 2015). Scoring of DAPI-stained cactin-depleted cells cactin but, unexpectedly, not of the core spliceosome component revealed nuclear morphology aberrations, with polylobate and SF3A-3 (Fig. 4E). By contrast, intron 5 was retained at similar levels fragmented nuclei progressively accruing over the experimental in cactin- and SF3A-3-depleted cells (Fig. 4E). Confirming the time course (Fig. 1E, Fig. 2E). Furthermore, metaphase specificity of our findings, UTR-less cactin expression alleviated chromosome spread analysis showed a prominent defect in sister sororin intron 1 retention in shCacD-transfected HEK 293T cells chromatid cohesion, with up to ∼90% of metaphases from depleted (Fig. S2F). Lastly, sororin intron 1 splicing was executed efficiently cells comprising prematurely separated and often hyper-condensed in different phases of the cell cycle (Fig. S2L,M), ruling out the sister chromatids (Fig. 3A,B). Taken together, these results reveal possibility that intron 1 retention in cactin-depleted cells is an that cactin is essential to support cellular proliferation, genome indirect consequence of G2/M arrest. Thus, cactin is essential for stability, proper nuclear morphology and chromosome segregation. splicing of sororin pre-mRNA and cactin depletion acutely affects All phenotypes are true outcomes of cactin deficiency because: splicing of intron 1. Retention of sororin intron 1 is expected to (1) they were induced by independent siRNAs, targeting different deplete functional sororin proteins in cells either resulting from a regions of cactin mRNA; (2) retrovirus-mediated overexpression of failure in mRNA export from the nucleus or, if the aberrant mRNA cactin from a UTR-less cDNA averted those phenotypes in U2OS is exported to the cytoplasm, from the introduction of a premature and in human embryonic kidney HEK 293T cells expressing a short stop codon that would allow translation of a truncated protein of 117 hairpin RNA (shRNA) targeting the same sequence as siCacD amino acids instead of 467 (Oka et al., 2014; Sundaramoorthy et al., (Fig. 3A,B; Fig. S2A-E). 2014; van der Lelij et al., 2014; Watrin et al., 2014). Indeed, western blot analysis revealed that the levels of full-length sororin were Cactin supports splicing of thousands of pre-mRNAs approximately halved in cactin-depleted samples when compared Data from different organisms suggest that cactin proteins function with control samples (Fig. 4F). in pre-mRNA splicing (An et al., 2013; Baldwin et al., 2013; As mentioned above, sororin is a cohesin-associated factor and Bessonov et al., 2008; Doherty et al., 2014; Ewing et al., 2007; Giot defects in sororin splicing strongly impair sister chromatid cohesion et al., 2003; Herold et al., 2009; Jurica et al., 2002; Lorenzi et al., (Oka et al., 2014; Sundaramoorthy et al., 2014; van der Lelij et al., 2015; Rappsilber et al., 2002; Zhou et al., 2002). To directly test the 2014; Watrin et al., 2014), as is also the case for cactin depletion. To requirement of cactin for pre-mRNA splicing, we deep-sequenced investigate to what extent sororin mis-splicing contributed to the poly(A)+ RNA from U2OS cells transfected with two cactin cohesion defects induced by cactin depletion, we depleted cactin in shRNA-expressing plasmids (shCacC and shCacD), as well as from HeLa cells infected with lentiviruses carrying an intron-less and shRNA-control-transfected cells. Both shRNAs were functional UTR-less sororin cDNA under the control of a doxycycline (dox)- as they efficiently depleted cactin and led to accumulation of inducible promoter (pL-Sor). As shown by northern blot, a short G2/M cells, abnormal nuclei and 53BP1 foci (Fig. S2G-I). sororin transcript corresponding to spliced and UTR-less sororin RNA sequencing analysis revealed a mild overall increase in the (herein referred to as ectopic RNA or eRNA), was readily induced relative abundance of introns in both depleted samples (Fig. 4A). by dox (Fig. 5A). Dox treatments did not affect endogenous sororin Specifically, a total of 32,661 introns from 7950 different genes intron 1 retention when cactin was depleted either in pL-Sor cells or were significantly differentially retained (adjusted P<0.1) in empty vector control cells (pL-ev; Fig. 5A). Expression of sororin samples depleted using both shRNAs as compared with samples eRNA almost completely averted premature sister chromatid from shRNA-control-transfected cells (Fig. S3A; Table S1). The separation (PSCS) and to a large extent abnormally shaped nuclei. vast majority of these (>91% in each case) showed increased By contrast, G2/M cell accumulation was only partly rescued and Journal of Cell Science

770 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068

Fig. 3. Cactin is required for sister chromatid cohesion. (A) Examples of DAPI-stained metaphase from the HeLa, U2OS or HEK 293T cells transfected with the indicated siRNAs or shRNAs. HEK 293T cells were previously infected with retroviruses carrying a 3′-UTR-less cactin cDNA (pB-Cac) or empty vector control retroviruses (pB-ev). Metaphase cells were collected 72 h post-transfection. White arrows point to the chromosomes enlarged in the insets. (B) Percentages of metaphases from the cells collected in A showing premature sister chromatid separation (PSCS). Data represented as mean±s.e.m from three independent experiments. At least 100 metaphases were scored for each sample in each experiment. ***P<0.001 (relative to siCtrl for Hela and U2OS, relative to shCtrl/pB-ev for HEK 293T; two-tailed Student’s t-test).

53BP1 focus frequencies were left essentially unchanged (Fig. 5B-D). analyzed sororin splicing by northern blot and RT-PCR (Fig. 6A,B). Hence, cactin supports normal chromosome cohesion and nuclear Despite variable efficiencies of siRNA-mediated depletion of target structure largely by promoting accurate splicing of sororin mRNAs (Fig. 6B), depletion of the ATPase/RNA helicase DEAH- pre-mRNA. box helicase 8 (DHX8) and the SR protein serine/arginine repetitive matrix 2 (SRRM2), both functional components of the spliceosome, Cactin physically and functionally interacts with DHX8 and stabilized intron-1-retaining sororin transcripts (Fig. 6A,B). SRRM2 Although we do not exclude that sub-optimal depletion of other To further shed light on the functions associated with cactin we tested factors might have not been sufficient to induce detectable decided to characterize its protein interactome, as this has not yet defects in sororin pre-mRNA splicing, we focused on DHX8 and been directly explored. We generated a HEK 293T cell line SRRM2 as our data suggested both physical and functional ectopically expressing a dox-inducible version of cactin interactions with cactin. N-terminally tagged with Strep–HA (SHA–cactin; Fig. S4A). We first validated our mass spectrometry results by performing Mass spectrometric analysis of proteins co-purified with SHA– coimmunoprecipitation experiments using antibodies against cactin identified a number of putative interactors participating in endogenous proteins and HEK 293T cellular extracts. DHX8 and, diverse cellular processes (Fig. S4B-C, Table S2). We depleted the albeit at low levels, SRRM2, were detected in protein fractions majority of the identified factors in HeLa cells using siRNAs and immunoprecipitated with anti-cactin antibodies, and cactin and Journal of Cell Science

771 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068

Fig. 4. Cactin promotes proper splicing of sororin pre-mRNA. (A-C) RNA-seq analysis of global pre-mRNA splicing efficiency in U2OS cells transfected with the indicated shRNA plasmids and harvested 48 h post-transfection. (A) The relative intron expression corresponds to the log-ratio between the estimated expression of introns and the average expression of exons in the corresponding gene. (B) Read coverage of the sororin gene in RNA-seq samples as in A. The sororin gene (CDCA5, RefSeq ID NM_080668) is sketched at the bottom with exons as blue boxes and introns as arrowed lines. The arrow points to reads corresponding to intron 1. (C) Relative expression of each sororin intron expressed as the ratio between the estimated expression of each intron and the average expression of exons in the sororin gene. FPKM, fragments per kilobase per million read counts. (D) Northern blot analysis of sororin transcripts using total RNA from HeLa cells transfected with the indicated siRNAs and harvested 72 h post-transfection. 18S serves as loading control. The two lowest panels show western blot analysis (west) of protein depletion. Actin serves as loading control. (E) RT-PCR analysis of sororin intron retention using total RNA from cells as in D. RNA from HeLa cells transfected with a shRNA against SF3A-3 was used as a control for global splicing inhibition. The oligonucleotides used to detect intron 1 and 5 are sketched at the bottom. (F) Western blot analysis of sororin levels in HeLa cells transfected with the indicated siRNAs and harvested 72 h post transfection. Actin serves as loading control. Numbers at the bottom correspond to sororin levels expressed as fold-decrease over the siCtrl sample after normalization to actin.

DHX8 coimmunoprecipitated with endogenous SRRM2 (Fig. S4D). depletion with a partly additive effect observed on accumulation This shows that cactin physically interacts with DHX8 and with of abnormal nuclei and G2/M cells when DHX8 and cactin were SRRM2, and that DHX8 physically interacts with SRRM2, possibly co-depleted (Fig. 6C-E). In SRRM2-depleted cells all examined pointing to the existence of cellular protein complexes comprising the aberrations accumulated at lower levels than in cactin- or DHX8- three factors. Our data also suggest that the interaction of SRRM2 depleted cells. No additive effect was observed when we with cactin-containing complexes might occur only transiently or at co-depleted SRRM2 and either cactin or DHX8 (Fig. 6C-E). low levels. We then analyzed the cellular distribution of the three Interestingly, depleting cactin or DHX8 strongly destabilized factors by indirect immunofluorescence following pre-extraction of SRRM2 total levels and accumulation within speckles (Fig. 6C,F); soluble material. All three proteins localized within the nucleus and depleting SRRM2 alone, and even more in combination with cactin, were excluded from the nucleolus. Cactin and DHX8 were evenly destabilized DHX8 total cellular levels (Fig. 6C); and depleting distributed within nuclei. SRRM2 formed prominent speckles SRRM2 in combination with DHX8 destabilized cactin total cellular generally devoid of the other two factors, but it was also diffused levels (Fig. 6C). We propose that cactin, DHX8 and SRRM2 form throughout the nucleus, covering regions containing cactin and functionally relevant cellular complexes supporting pre-mRNA DHX8 (Fig. S4E,F). Thus, interactions between the three factors splicing and that complex assembly contributes towards stabilizing could conceivably happen in the nucleus. the levels of the three factors within cells. Notably, DHX8 was Finally, we depleted cactin, DHX8 and SRRM2 either singly or previously found to interact with cactin in a high-throughput study in different combinations in HeLa cells and analyzed sororin (Huttlin et al., 2015), and MOG-5, the C. elegans ortholog of DHX8, splicing, PSCS, 53BP1 foci, abnormal nuclei and cell cycle was previously isolated as a component of the CACN-1 interactome progression. DHX8 depletion largely recapitulated cactin (Doherty et al., 2014). These data further corroborate our results and Journal of Cell Science

772 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068

Fig. 5. Expression of an intron-less sororin cDNA restores the defects associated with cactin depletion to different extents. HeLa cells were infected with lentiviruses carrying an intron-less, UTR-less sororin cDNA under the control of a doxycycline (dox)-inducible promoter (pL-Sor), and transfected with the indicated siRNAs. pL-ev, empty vector control lentivirus. Cells were harvested 72 h post-transfection and/or dox induction. (A) Northern blot analysis of sororin transcripts using total RNA from the treated HeLa cells. eRNA, ectopically expressed cDNA. 18S serves as loading control. The two lowest panels show a western blot analysis of protein depletion. Lamin B1 (LMB1) serves as loading control. (B) Quantifications of the indicated aberrations in the treated HeLa cells. Data represented as mean±s.e.m from three independent experiments. At least 100 cells were scored for each sample in each experiment. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (relative to pL-ev, siCtrl or no dox treatments; two-tailed Student’s t-test). (C) FACS analysis of treated HeLa cells infected with pL-Sor and stained with propidium iodide. (D) Examples of DAPI stained metaphase chromosomes from HeLa cells treated as indicated. suggest that cactin–DHX8 interactions are conserved throughout cactin has recently been identified as an RNA binding protein in a evolution. large-scale study where protein-RNA photocrosslinking was combined with quantitative mass spectrometry (He et al., 2016). DISCUSSION Impaired splicing of pre-mRNAs coding for factors involved in Using a protein depletion approach, we have shown that cactin is different cellular processes could already explain the diversity of the essential for normal physiology of human cells. Moreover, defects arising upon cactin inactivation. We have demonstrated that consistent with previous reports implicating cactins in pre-mRNA complete splicing of sororin pre-mRNA is sufficient to largely splicing, we find that cactin promotes faithful splicing of thousands explain how cactin promotes proper chromosome separation and, to of different transcripts. The exact mechanism by which cactin some extent, cell cycle progression. Aberrant splicing of transcripts promotes splicing will be the subject of future investigations; involved in suppressing DNA damage accumulation or in DNA nevertheless, its physical interaction with DHX8 and SRRM2, and damage repair could explain why cactin-depleted cells show marks other members of the spliceosome machinery from different of damaged DNA. Consistently, accumulating evidence indicates organisms (Bessonov et al., 2008; Doherty et al., 2014; Ewing that in the absence of functional splicing cells are unable to properly et al., 2007; Giot et al., 2003; Herold et al., 2009; Jurica et al., 2002; repair broken DNA. For example, genome-wide siRNA screens Rappsilber et al., 2002; Zhou et al., 2002), suggests that it is a have shown that depletion of various factors involved in splicing component of the active spliceosome. Prp22p and Cwc21p, the and RNA processing impairs DNA double-strand break repair, thus putative budding yeast orthologs of DHX8 and SRRM2, inducing genomic instability (Adamson et al., 2012; Paulsen et al., respectively, participate in the selection of intronic 3′ end splice 2009). Moreover, splicing promotes proper function of the E3 sites (3′ss) (Gautam et al., 2015; Schwer and Gross, 1998; Semlow ubiquitin ligase RNF8 at sites of DNA damage (Pederiva et al., et al., 2016). It is conceivable that erroneous 3′ss selection could 2016). According to our transcriptome analysis, several transcripts occur when cactin is depleted. Moreover, the helicase activity of encoding factors supporting genome stability are mis-spliced when Prp22p is required for proper release of mature mRNAs after cactin is depleted. Amongst these, introns from RAD50, RAD52, completion of the splicing reaction (Company et al., 1991; Schwer, UPF1, RIF1 and RNF8 itself, all involved in maintaining genome 2008; Schwer and Gross, 1998), suggesting that cactin could also integrity, are over-represented in libraries from cactin-depleted cells participate in spliceosome disassembly and mRNA export. Further (see Table S1). In a similar manner to the approach that we have supporting a direct role for cactins in pre-mRNA splicing, mouse used for sororin, expression of intron-less cDNAs for each of these Journal of Cell Science

773 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068

Fig. 6. Cactin physically and functionally interacts with DHX8 and SRRM2. (A,B) HeLa cells were transfected with the indicated siRNAs and harvested 72 h post-transfection. (A) Northern blot analysis with 18S as loading control. (B) RT-PCR analysis of sororin intron 1 retention using total RNA of sororin transcripts. Numbers at the bottom indicate the fraction of remaining targeted mRNA as determined by quantitative RT-PCR and expressed as percentage of mRNA in siCtrl- transfected cells. (C-E) HeLa cells were transfected with the indicated siRNAs and harvested 72 h post-transfection. (C) Western blot, northern blot and RT-PCR analysis of cells with LMB1 and 18S serving as loading controls for proteins and RNA, respectively. The ratios between intensities of PCR products corresponding to intron-1-retaining and spliced sororin mRNAs are indicated at the bottom of the RT-PCR panel. (D) Quantifications of the indicated aberrations in cells. Data represented as mean±s.e.m from three independent experiments. At least 100 nuclei or 30 metaphases were scored for each sample in each experiment. *P<0.05, **P<0.01, ***P<0.001 (relative to pL-ev, siCtrl or no dox treatments; two-tailed Student’s t-test). (E) FACS analysis of treated HeLa cells stained with propidium iodide. (F) Indirect immunofluorescence analysis of HeLa cells transfected with the indicated siRNAs. Cells were harvested 72 h post transfection. factors in combination with cactin depletion will help in (Stracker and Petrini, 2011). It is therefore possible that cactin understanding to what extent they are involved in the DNA deficiency causes genome instability not only by affecting splicing damage response arising when cactin is depleted. but also by directly interfering with MRN functions. However, our characterization of the protein interactome of We have also found that cactin physically interacts with CUL7 cactin suggests the intriguing and not mutually exclusive possibility (Fig. S4C, Table S2), a large E3 ubiquitin ligase conserved only in that the pleiotropy of cactin inactivation defects derives from vertebrates. CUL7 forms the so-called 3M complex together with concomitant impairment of pathways that do not intersect with pre- the two other core components, OBSL1 and CCDC8. Mutually mRNA splicing. We found that cactin physically interacts with exclusive mutations in one of the three genes are associated with the RAD50 and Mre11 (Fig. S4C, Table S2), which form the MRN 3M syndrome, a rare hereditary disorder characterized by severe pre- complex together with NBS1. MRN supports genome stability by and post-natal growth retardation (Clayton et al., 2012). The 3M mediating DNA double-strand break repair and DNA recombination complex also interacts with several additional proteins, including Journal of Cell Science

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FBXW8, a substrate adaptor for CUL7 (Yan et al., 2014). OBSL1, cycle synchronization, cells were treated for 20 h with 2 mM thymidine CCDC8 and FBXW8 were all identified in our cactin interactome (Sigma-Aldrich) and then released into normal medium. 4 h after release, (Table S2), making cactin a possible component of the 3M complex. cells were incubated with 100 ng/ml nocodazole for 12 h, collected by In this regard, cactin was not identified in a mass-spectrometry- mitotic shake-off and released into normal medium. shRNA-expressing based study where CUL7, OBSL1 and CCDC8 were over-expressed plasmids were generated in pSUPER-Puro (Azzalin and Lingner, 2006) and transfected using Lipofectamine 2000 reagent (Invitrogen), followed by in HEK 293 cells and used as bait (Hanson et al., 2014). Because we selection with 1 µg/ml puromycin (Sigma-Aldrich). mRNA target sequen- performed our experiments in HEK 293T cells (HEK 293 cells ces were as follows: 5′-GCTTCGAGTGGAACAAGTACAAC-3′ (shCacC); stably expressing the SV40 large T antigen), it is possible that the 5′-ATGGAATTGGCCTATTGGCAAGA-3′ (shCacD); 5′-GGAAATAGT- SV40 large T antigen, which is known to associate with CUL7 and TCCGTTTGTTTCTC-3′ (shCacE); 5′-GGAGGAGCTCAATGCCATT-3′ inhibit its ubiquitin ligase activity (Hartmann et al., 2014), (shSF3A-3). siRNAs were purchased from Qiagen and transfected at 20– stimulates cactin interaction with the 3M complex. Alternatively, 50 nM using Lipofectamine RNAiMAX (Invitrogen). siRNAs were as foll- cactin might be associated with the other elements of the 3M ows: siCtrl (#1027310); siCacD (5′-ATGGAATTGGCCTATTGGCAA-3′); complex only transiently or at very low levels and therefore escaped siCacE (5′-AAATAGTTCCGTTTGTTTCTC-3′); siCUL7 (#SI00357000); α detection in experiments where CUL7, OBSL1 and CCDC8 were siOBSL1 (#SI04339951); siCCDC8 (#SI05077086); siCK2 (#SI02660504); used as bait (Hanson et al., 2014). Because the 3M complex sicXorf56 (#SI03195731); siUBR5 (#SI03074225); siUSP9X (#SI00066584); siDDX1 (#SI00299978); siDHX8 (#SI03019373); siSRRM2 (#SI04173995); supports microtubule and genome integrity (Yan et al., 2014), the siSRPK1 (#SI02223109). genomic instability and cell proliferation defects observed in cells depleted for cactin could be linked to impaired 3M complex Streptavidin purification for mass spectrometry analysis function. Our data also imply the possible existence of so-far- Approximately 1×108 Flp-In T-Rex 293 cells expressing HA–Strep-tagged unexplored links between cactin and developmental disorders or cactin were harvested and resuspended in ice-cold HNN lysis buffer [50 mM between the 3M syndrome and pre-mRNA splicing defects. HEPES, 150 mM NaCl, 50 mM NaF, 0.5% NP-40, 1 mM PMSF, 1× Although preliminary experiments did not reveal defects in phosphatase and 1× protease inhibitor cocktails (Sigma-Aldrich), 10 µg/ml sororin intron 1 splicing when CUL7 was siRNA-depleted Avidin (IBA)]. After 10 min incubation on ice, lysates were centrifuged at (Fig. 4D,E and data not shown), the latter hypothesis is consistent high speed and supernatants were loaded on Bio-Spin chromatography with the enrichment of several pre-mRNA splicing factors in the columns (Bio-Rad) containing 200 µl of 50% slurry Strep-Tactin sepharose CUL7, OBSL1 and CCDC8 interactome (Hanson et al., 2014). beads (IBA) equilibrated in HNN lysis buffer. Beads were washed twice with HNN lysis buffer and three times with HNN buffer (50 mM HEPES, Overall, cactin seems to be a versatile, multifunctional player 150 mM NaCl, 50 mM NaF) followed by elution with 0.5 mM Biotin participating in several cellular processes essential for cell viability (Sigma-Aldrich) in HNN buffer. Eluted proteins were boiled in 2× Laemmli and/or proliferation, and chromosome stability at large. Our study, buffer at 95°C for 5 min and loaded onto gradient polyacrylamide gels besides corroborating the essentiality of cactin for cell physiology (Invitrogen). Gels were fixed and stained with AgNO3 according to standard and its direct connections with pre-mRNA splicing, expands our protocols and bands were excised and sent for mass-spectrometry-based understanding of the broad spectrum of cellular and organismal analysis to Alphalyse (Denmark). defects caused by cactin deficiency across eukaryotes. Immunoprecipitation MATERIALS AND METHODS Approximately 5×106 cells were harvested and resuspended in ice-cold lysis Plasmids buffer [50 mM Tris pH 7.4, 1.5 mM MgCl2, 1 mM EDTA, 150 mM NaCl, A plasmid containing full-length cactin (C19orf29) cDNA was purchased 0.5% Triton X-100, 1 mM DTT, 1× phosphatase and 1× protease inhibitor from Origene and utilized for successive plasmid constructions. pB-Cac and cocktails (Sigma-Aldrich)] followed by incubation on ice for 30 min. pSHA-Cac were generated by ligating an N-terminally HA–Strep-tagged Supernatants were recovered by high-speed centrifugation and pre-cleared cactin cDNA into the pBABE-Hygro and the pcDNA5/FRT/TO-Hygro by incubation with 20 µl of 50% slurry Protein A/G agarose beads (Santa Cruz (Life Technologies) plasmids, respectively. pL-Sor was generated by Biotechnology) at 4°C on a wheel. Beads were eliminated by high-speed ligating sororin cDNA PCR-amplified from reverse-transcribed HeLa total centrifugation and 500 µg of proteins were incubated with 4 µg of primary RNA into the pLVX-Puro vector (Clontech). antibodies for 4 h at 4°C. Antibodies were as follow: mouse monoclonal anti- HA (Santa Cruz Biotechnology, sc-57592); mouse monoclonal anti-Myc (Cell Cell lines and tissue culture procedures Osteosarcoma Signaling, 2276); rabbit polyclonal anti-cactin (Bethyl Laboratories, A303- Osteosarcoma U2OS (a kind gift from Massimo Lopes, IMCR, Zürich, 349A); rabbit polyclonal anti-CUL7 (GeneTex, GTX113906); mouse Switzerland), cervical carcinoma HeLa (ATCC), human embryonic kidney monoclonal anti-SRRM2 (Santa Cruz Biotechnology, sc390315). Lysates expressing SV40 large T antigen HEK 293T (ATCC) and Flp-In T-Rex 293 were incubated with 30 µl of 50% slurry Protein A/G beads for ∼16 h at 4°C (Thermo Fisher Scientific) cells were cultured in high-glucose D-MEM on a rotating wheel. Beads were washed three times with IP buffer (50 mM Tris (Invitrogen) supplemented with 10% TET-free FBS (Pan Bio Tech) and pH 7.4, 1.5 mM MgCl2, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100), 100 U/ml penicillin–streptomycin (Sigma-Aldrich). All cell lines were once with wash buffer (50 mM Tris pH 7.4, 1.5 mM MgCl2, 1 mM EDTA, routinely tested for bacteria and mycoplasma contaminations. For 150 mM NaCl) and then boiled in 2× Laemmli buffer for 95°C for 5 min. constitutive expression of ectopic cactin, U2OS and HEK 293T cells were infected with retroviruses prepared according to standard protocols using Western blotting pB-Cac, followed by selection with 200 µg/ml Hygromycin B (Fluka). For Western blot analysis was carried out according to standard procedures inducible expression of ectopic cactin, Flp-In T-Rex 293 cells were using the following antibodies: rabbit polyclonal anti-cactin (Abnova, transfected with pSHA-Cac, followed by selection with 200 µg/ml Zeocin PAB23952, diluted 1:1000); rabbit polyclonal anti-sororin (kind gift from (Invitrogen). For inducible expression of ectopic sororin, HeLa cells were Jan-Michael Peters, IMP, Vienna, Austria; 1:1000); rabbit polyclonal anti- infected with lentiviruses prepared according to standard protocols using UPF1 (Chawla et al., 2011) (raised against a peptide corresponding to the pL-Sor, followed by selection with 1 µg/ml puromycin (Sigma-Aldrich) and UPF1 C-terminal sequence ERAYQHGGVTGLSQY at PolyPeptide clonal cell line isolation. For transcription induction, cells were treated with Group; 1:1000); mouse monoclonal anti-golgin 97 (Molecular Probes, A- 500 ng/ml doxycycline (Sigma-Aldrich). For DNA damage induction, cells 21270, 1:1000); rabbit polyclonal anti-lamin-B1 (GeneTex, GTX103292, were treated with 5 µg/ml bleomycin (Sigma-Aldrich) for 2 h or with 1:1000); mouse monoclonal anti-actin (Abcam, ab8224, 1:5000); rabbit 100 nM camptothecin (Sigma-Aldrich) for 12 h or 500 nM for 4 h. For cell polyclonal anti-DHX8 (Bethyl Laboratories, A300-624A-M, 1:1000); Journal of Cell Science

775 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 767-778 doi:10.1242/jcs.194068 mouse monoclonal anti-SRRM2 (Santa Cruz Biotechnology, sc-390315, Invitrogen) was added to cultured cells for 30 min before harvesting. Mouse 1:200); mouse monoclonal anti-TRF2 (Millipore, 05-521, 1:1000); mouse monoclonal anti-γH2AX (Millipore, 05-636, 1:500), Click-It reaction mix monoclonal anti-TRF1 (Abcam, ab10579, 1:1000); rabbit polyclonal anti- containing Alexa Fluor 488 Azide (Invitrogen) and DAPI (1 µg/ml, Sigma- RAP1 (Bethyl Laboratories, A300-306A, 1:500); mouse monoclonal anti- Aldrich) were used. Stained cells were sorted and analyzed using a Fortessa TPP1 (Abnova, H0006557-M02, 1:500); mouse monoclonal anti-HA flow cytometer (BD Bioscience) and FlowJo software. (Santa Cruz Biotechnology, sc-57592, 1:2000); mouse monoclonal anti- Myc (Cell Signaling, 2276, 1:2000); rabbit polyclonal anti-KAP1 (Bethyl Pulse field gel electrophoresis (PFGE) analysis of DNA double- Laboratories, A300-274A, 1:2000); rabbit polyclonal anti-phospho-KAP1 strand breaks S824 (Bethyl Laboratories, A300-767A, 1:1000); mouse monoclonal anti- PFGE analysis was performed according to previously published protocols CHK1 (Santa Cruz Biotechnology, sc-8408, 1:1000); rabbit monoclonal (Toller et al., 2011). Briefly, cells were harvested and embedded in 1% anti-phospho-CHK1 S345 (Cell Signaling, 2348, 1:500); rabbit polyclonal molten LMT agarose (Bio-Rad). Solidified plugs were incubated at 37°C anti-phospho-RPA32 (S33) (Bethyl Laboratories, A300-246A, 1:1000); for 36–72 h in lysis buffer (100 nM EDTA pH 8.0, 0.2% sodium HRP-conjugated goat anti-mouse and anti-rabbit IgGs (Bethyl Laboratories, deoxycholate, 1% sodium lauryl sarcosine) containing Proteinase K A90-116P and A120-101P, 1:5000). Signals were acquired using (1 mg/ml, Appli Chem). Plugs were embedded into a 0.9% pulse-field- FluorChem HD2 apparatus (Alpha Innotech). certified agarose gel (Bio-Rad) and subjected to pulse-field electrophoresis in a CHEF DR III apparatus (Bio-Rad) at 14°C, stained Indirect immunofluorescence, DNA fluorescence in situ with ethidium bromide (0.5 µg/ml) and imaged using a Gel Doc 2000 hybridization (FISH) and metaphase spread analysis imaging system (Bio-Rad). Indirect immunofluorescence analysis was carried out according to standard protocols. For indirect immunofluorescence and FISH, cells grown on RNA analysis coverslips were subjected to pre-extraction in CSK buffer (100 mM NaCl, Total RNA was isolated using a Nucleo Spin RNA Kit Plus (Macherey- 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES pH 6.8, 0.5% Triton-X) for Nagel) or TRIzol reagent (Invitrogen) and treated at least once with DNaseI 7 min on ice, fixed with 2% formaldehyde for 10 min at room temperature, (Qiagen) according to manufacturer’s instructions. For RT-PCR, 1 µg of permeabilised with 0.5% Triton X-100 for 10 min at room temperature, and RNA was reverse-transcribed using Superscript II reverse transcriptase re-fixed with methanol for 20 min at −20°C. Fixed cells were incubated in (Invitrogen) and random hexamers followed by PCR amplification using blocking solution (5% BSA, 20 mM glycine, 1× PBS) supplemented with Taq polymerase (New England Biolabs) or real-time PCR amplification RNaseA (20 µg/ml, Roche) for 30 min at 37°C, followed by incubation with using the Light Cycler 498 SYBR Green I master mix (Roche) on a Rotor- primary and then secondary antibodies diluted in 5% BSA, 1× PBS at room Gene Q instrument (Qiagen) using actin mRNA as normalizer. temperature for 1 h each. Cells were again fixed with 4% formaldehyde for Oligonucleotide pairs are shown in Table S3. For northern blot analysis, 10 min at room temperature and DNA was denatured by incubating 10-15 µg of RNA were denatured at 65°C for 5 min and subjected to coverslips in hybridization mix [10 mM Tris pH 7.2, 70% formamide, 0.5% electrophoresis in denaturing 1.2% agarose gels. RNA was transferred onto blocking solution (Roche)] containing telomeric or centromeric PNA probes nylon membranes and hybridized using radioactively labeled probes for for 5 min at 80°C on a heating plate. Hybridizations were carried out for 2 h 16 h at 50–60°C. The sororin probe was a random-primer-labeled PCR at room temperature and were followed by two washes with wash 1 solution amplification product from the pL-Sor plasmid. The 18S rRNA probe was a (10 mM Tris pH 7.2, 70% formamide, 0.1% BSA) and one with wash 2 5′-end-labeled oligonucleotide. Radioactive signals were detected using a solution (0.1 M Tris pH 7.2, 0.15 M NaCl, 0.08% Tween-20) for 10 min Typhoon FLA 9000 imager (GE Healthcare). For transcriptome analysis, each at room temperature. DNA was counterstained with DAPI (100 ng/ml, 5 µg of RNA were used for library preparation and high-throughput Illumina Sigma-Aldrich). Antibodies used for indirect immunofluorescence sequencing at Fasteris SA (Plan-les-Ouates, Switzerland). Sequencing was analysis were: rabbit polyclonal anti-cactin (Abnova, PAB23952, diluted performed with a single-end protocol providing reads of 100 bp in length. 1:1000); rabbit polyclonal anti-53BP1 (Abcam, ab21083, 1:1000); rabbit Reads were trimmed with sickle software (available from https://github.com/ polyclonal anti-DHX8 (Bethyl Laboratories, A300-624A-M, 1:1000); najoshi/sickle) and aligned to the human reference genome (https://www. mouse monoclonal anti-SRRM2 (Santa Cruz Biotechnology, sc-390315, gencodegenes.org; GENCODE GRCh37 v19) using HISAT2 software 1:1000); rabbit polyclonal anti-phospho-RPA32 S33 (Bethyl Laboratories, (v2.0.4) (Kim et al., 2015). DEXSeq (v1.20.0) (Anders et al., 2012) was A300-246A, 1:1000). PNA FISH probes were: 5′-Cy3-OO- used to investigate the presence of intron retention. From the GENCODE CCCTAACCCTAACCCTAA-3′ (TelC-Cy3, Panagene); 5′-FAM- catalog, we retained exons from known, protein-coding, non-automatically AAACTAGACAAGCATT-3′ (Cent-FAM, PNAbio). For metaphase annotated transcripts, and processed the resulting GTF file with the Python spread analysis, cells were treated with 200 ng/ml colcemid (Sigma- scripts provided with DEXSeq in order to generate disjoint exon bins. The Aldrich) for 2–4 h, harvested by mitotic shake-off and incubated in ‘flattened’ annotation was extended with intron bins, defined as intragenic hypotonic solution (0.075 M KCl) for 9 min at 37°C. Metaphase regions disjoint from any retained exon. DEXSeq was used to quantify the chromosomes were resuspended in cold methanol:acetic acid solution abundance of each exonic and intronic bin in this extended annotation, and to (3:1) by vortexing and incubated overnight at −20°C. Metaphases were test each bin for differential usage between the control group and each shRNA- spread on glass slides and counterstained with DAPI (100 ng/ml, Sigma- transfected group. Only results for intronic bins were kept for interpretation. A Aldrich). Images were acquired with an Olympus IX 81 microscope fragments per kilobase per million mapped reads (FPKM) estimate was equipped with a Hamamatsu ORCA-ER camera using a 60×1.42NA oil generated for each intronic and exonic bin by dividing the counts obtained from PlanAPoN objective, or with a Deltavision Multiplexed system (Applied DEXSeq (after adding 1) by the size of the bin and the total number of read Precision) with an Olympus 1X 71 (inverse) microscope, Roper CoolSnap counts for the corresponding sample. Relative intron expression was calculated HQ2 camera and a 60×1.4NA oil DIC PlanAPoN objective. Images were for each intron bin by dividing its estimated expression with the average analyzed using ImageJ (NIH) and Adobe Photoshop. expression of the exon bins from the same gene. Alignment files generated by HISAT2 were filtered to retain only reads with mapping quality above 10 and Flow cytometric analysis used to visualize the coverage of selected genes with the Gviz R package Cells were fixed in ice-cold 70% ethanol for 15 min at −20°C, and then (v 1.18.0) (Hahne and Ivanek, 2016). enrichment analysis was incubated with 25 µg/ml RNaseA (Sigma-Aldrich) diluted in 1× PBS for performed using the PANTHER (protein analysis through evolutionary 20 min at 37°C. Cells were stained with 20 µg/ml propidium iodide (Sigma- relationships) classification system (http://www.pantherdb.org). Aldrich) for 10 min at room temperature, sorted and analyzed with a BD FACSCalibur (BD Biosciences) using FlowJo software. Flow cytometric Error and statistical calculations analysis for DNA synthesis rates and antibody staining was performed Samples sizes (n) were derived from experiments with independent cell according to previously published protocols (Neelsen et al., 2013). 10 µM cultures. Experimental data are presented as the mean±s.e.m. Statistical halogenated nucleotide EdU (Click-IT EdU Flow Cytometry Assay Kit, calculations were performed in Microsoft Excel. Journal of Cell Science

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Acknowledgements Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y. L., We thank Jan-Michael Peters, Massimo Lopes, Ulrike Kutay (IBC, Zürich, Ooi, C. E., Godwin, B., Vitols, E. et al. (2003). A protein interaction map of Switzerland) for reagents; Harry Wischnewski, Catherine Brun and Federica Richina Drosophila melanogaster. Science 302, 1727-1736. for help with experiments; Rajika Arora for critical reading of the manuscript; Hahne, F. and Ivanek, R. (2016). Visualizing genomic data using Gviz and members of the Azzalin laboratory for discussions. bioconductor. Methods Mol. Biol. 1418, 335-351. Hanson, D., Stevens, A., Murray, P. G., Black, G. C. M. and Clayton, P. E. (2014). Identifying biological pathways that underlie primordial short stature using network Competing interests analysis. J. Mol. Endocrinol. 52, 333-344. The authors declare no competing or financial interests. Hartmann, T., Xu, X., Kronast, M., Muehlich, S., Meyer, K., Zimmermann, W., Hurwitz, J., Pan, Z.-Q., Engelhardt, S. and Sarikas, A. (2014). Inhibition of Author contributions Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen. Proc. Natl. I.M.Y.Z., L.E.L. and C.M.A. designed the experiments. I.M.Y.Z. and L.E.L. performed Acad. Sci. USA 111, 3371-3376. the experiments and analyzed the data. C.S. performed the analysis of next He, C., Sidoli, S., Warneford-Thomson, R., Tatomer, D. C., Wilusz, J. E., Garcia, generation sequencing data. I.M.Y.Z. and C.M.A. wrote the manuscript. B. A. and Bonasio, R. (2016). High-resolution mapping of RNA-binding regions in the nuclear proteome of embryonic stem cells. Mol. Cell 64, 416-430. Funding Herold, N., Will, C. L., Wolf, E., Kastner, B., Urlaub, H. and Luhrmann, R. (2009). This work was supported by the European Research Council (BFTERRA) and the Conservation of the protein composition and electron microscopy structure of Mol. Cell. Biol. Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung Drosophila melanogaster and human spliceosomal complexes. 29, (Swiss National Science Foundation) (31003A_160338). 281-301. Huttlin, E. L., Ting, L., Bruckner, R. J., Gebreab, F., Gygi, M. P., Szpyt, J., Tam, S., Zarraga, G., Colby, G., Baltier, K. et al. (2015). The BioPlex Network: a Data availability systematic exploration of the human interactome. Cell 162, 425-440. ’ RNA sequencing data have been deposited in NCBI s Gene Expression Omnibus Jurica, M. S., Licklider, L. J., Gygi, S. R., Grigorieff, N. and Moore, M. J. (2002). and are accessible through GEO Series accession number GSE82070 (https://www. Purification and characterization of native spliceosomes suitable for three- ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE82070). dimensional structural analysis. RNA 8, 426-439. Kim, D., Langmead, B. and Salzberg, S. L. (2015). 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