© 2019. Published by The Company of Biologists Ltd | Journal of Science (2019) 132, jcs232728. doi:10.1242/jcs.232728

RESEARCH ARTICLE Role for the splicing factor TCERG1 in Cajal body integrity and snRNP assembly Cristina Moreno-Castro1, Silvia Prieto-Sánchez1, Noemı́Sánchez-Hernández1, Cristina Hernández-Munain2 and Carlos Suñé1,*

ABSTRACT 2016). Nuclear processing of is performed in the Cajal Cajal bodies are nuclear involved in the nuclear phase of bodies (CBs). After the final maturation process, fully assembled small nuclear ribonucleoprotein (snRNP) biogenesis. In this study, we snRNPs are released from CBs to the nuclear speckles, where they identified the splicing factor TCERG1 as a -associated factor accumulate prior to functioning in the splicing process. that is essential for Cajal body integrity. Knockdown of TCERG1 CBs were first discovered in vertebrate by Spanish disrupts the localization of the components of Cajal bodies, including neurobiologist Santiago Ramón y Cajal in 1903 (Cajal, 1903). coilin and NOLC1, with coilin being dispersed in the into Despite having been described more than a century ago, the major numerous small foci, without affecting speckles, gems or the molecular components and functions of CBs related to snRNP locus body. Furthermore, the depletion of TCERG1 affects the assembly and recycling have only begun to be deciphered over the recruitment of Sm to uridine-rich small nuclear past 20 years (Gall, 2000). It has been demonstrated that a complex (snRNAs) to form the mature core snRNP. Taken together, the termed the little elongation complex (LEC) facilitates efficient results of this study suggest that TCERG1 plays an important role in synthesis of snRNA by RNAPII (Hu et al., 2013; Smith et al., 2011). Cajal body formation and snRNP biogenesis. In mammalian cells, LEC is composed of ELL, ICE1, and ICE2 proteins, which colocalize with coilin in CBs (Hu et al., 2013; Polak KEY WORDS: Cajal body, snRNPs, TCERG1, Coilin, Splicing et al., 2003; Smith et al., 2011). Recently, Hutten and collaborators found that the SUMO isopeptidase USPL1, which is an essential INTRODUCTION component of CBs, interacts and colocalizes with components Nuclear precursor mRNA (pre-mRNA) splicing is a fundamental of the LEC. Importantly, knockdown of USPL1 resulted in RNA processing mechanism that occurs as a common means of disassembly of CBs, reduced RNAPII-mediated snRNP achieving proteomic cell diversity. Global transcriptome analyses and altered pre-mRNA splicing (Hutten et al., 2014). estimate that more than 90% of human multiexon genes undergo More recently, the human 7SK snRNP, composed of the 7SK alternative splicing (Pan et al., 2008; Wang et al., 2008). Splicing is snRNA Larp7 and MePCE, has also been found to specifically catalyzed by a dynamic ribonucleoprotein machinery known as the associate with the LEC to promote RNAPII-mediated snRNA and spliceosome, which is composed of RNA– complexes called small nucleolar RNA (snoRNA) synthesis (Egloff et al., 2017). small nuclear ribonucleoproteins (snRNPs) and a group of ∼200 These data, together with results showing that the human mediator additional proteins (Matera and Wang, 2014; Wahl et al., 2009). The subunit MED26 also localizes to CBs and plays a crucial role in the snRNPs are composed of five uridine (U)-rich small nuclear RNAs transcription of snRNA genes through recruitment of the LEC (snRNAs), U1, U2, U4, U5 and U6, as well as a heptameric ring of (Takahashi et al., 2015), support the view that CBs have a role in Sm proteins and a specific set of proteins. Spliceosomal snRNP snRNP and snoRNP biogenesis. Further evidence has been found formation undergoes an intricate assembly and maturation pathway for the role of CBs in snRNA processing. The RNAPII-associated that involves nuclear and cytoplasmic events. After transcription and integrator complex (INT) is required for the integrity of CBs and for ′ initial 3′ end processing, snRNAs are exported to the cytoplasm, cleaving the extended 3 -end of snRNAs, which is essential for the where the heptameric ring of Sm proteins is assembled followed by biogenesis of spliceosomal snRNAs (Albrecht et al., 2018; Takata hypermethylation of the 7-methylguanosine (m7G) snRNP cap. et al., 2012). The target of EGR1 protein 1 (TOE1) is a nuclear ′ Then, the resulting immature snRNPs return to the nucleus, where deadenylase that localizes to CBs and participates in the 3 -end additional snRNP-specific proteins are added to assemble functional processing of small Cajal body-specific RNAs (scaRNAs), di-snRNP (U4 and U6) and tri-snRNP complexes (U4, U6 and U5) snoRNAs, snRNAs and RNA component (TERC) in addition to snRNP recycling, as well as posttranscriptional (Lardelli et al., 2017; Son et al., 2018). In addition to being involved modifications (Fischer et al., 2011; Matera and Wang, 2014; Stanek,̌ in snRNP biogenesis, CBs also contain factors involved in other cellular processes, such as transcription and RNA stability, ribosome biogenesis, histone mRNA processing and 1Department of Molecular Biology, Institute of Parasitology and Biomedicine maintenance (Machyna et al., 2013). Many of these factors are “López-Neyra” (IPBLN-CSIC), PTS, 18016 Granada, Spain. 2Department of Cell not CB specific and are also present in other types of nuclear Biology and Immunology, Institute of Parasitology and Biomedicine “López-Neyra” bodies, such as gems, the histone locus body or the . (IPBLN-CSIC), PTS, 18016 Granada, Spain. A comprehensive list of relevant protein components of CBs has *Author for correspondence ([email protected]) recently been published (Sawyer et al., 2019). Coilin is a phosphoprotein that is commonly used to define CBs C.M.-C., 0000-0002-7172-7341; S.P.-S., 0000-0001-7950-8162; N.S.-H., 0000- 0002-1697-2352; C.H.-M., 0000-0002-6058-2458; C.S., 0000-0002-7991-0458 and is considered an essential structural component of these bodies because loss of coilin results in CB disintegration (Collier et al.,

Received 3 April 2019; Accepted 11 October 2019 2006; Liu et al., 2009; Strzelecka et al., 2010b; Tucker et al., 2001). Journal of Cell Science

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Although coilin appears not to be fully essential for viability in overexposure, we found coilin dispersed throughout the Arabidopsis, Drosophila or mice, coilin gene disruption provokes nucleoplasm, appearing as tiny dots in the nucleoplasm (Fig. 1A, embryonic lethality in zebrafish (Collier et al., 2006; Liu et al., overexposed panels), which is reminiscent of a phenotype resulting 2009; Tucker et al., 2001; Walker et al., 2009). Coilin depletion in from the depletion of CB components (Girard et al., 2006; Lemm zebrafish leads to defects in snRNP biogenesis that can be rescued et al., 2006; Li et al., 2014). Quantification analysis of CBs showed upon snRNP injection (Strzelecka et al., 2010b), which strongly a reduction in the number and size of these organelles in the suggests that CBs are essential for snRNP assembly. These data, TCERG1-knockdown HEK293T cells (Fig. 1B). Similar results together with other extensive studies that show accumulation of were obtained with the TCERG1-depleted HAP1 (Fig. 1A,B) or stalled intermediates upon depletion of snRNP-specific proteins or SH-SY5Y (Fig. S1) cells. Interestingly, knockdown of TCERG1 blockage of di- and tri-snRNP formation (Bizarro et al., 2015; did not change the total protein (Fig. 1C) or mRNA (Fig. 1D) levels Novotný et al., 2015; Schaffert et al., 2004) and the de novo CB of coilin. The use of a proximal start site of transcription in HAP1 formation triggered by inhibition of tri-snRNP (Novotný et al., cells and the difficulty in targeting each allele in the HEK293T 2015), suggest a role for CBs in quality control of snRNP assembly polyploidy cell line may account for the residual TCERG1 and maturation. This putative role for CBs is supported by expression in these cells (Fig. 1C). computational modeling, which predicts that the presence of Next, we assessed whether other components of CBs, which are these organelles increases the local concentration of snRNP shared with other known types of , are affected upon components, thereby enhancing snRNP assembly by up to 11-fold TCERG1 knockdown. NOLC1 is a nucleolar protein that shuttles (Klingauf et al., 2006). This predicted assembly rate was between the nucleolus and the cytoplasm, interacts with coilin and later experimentally verified by measuring the in vivo kinetics of accumulates at the CBs, suggesting that NOLC1 functions as a tri-snRNP assembly using fluorescent recovery after molecular link between the nucleolus and the CBs (Isaac et al., photobleaching (FRAP) (Novotný et al., 2011). The assembly 1998). Immunofluorescence studies showed that NOLC1 localized rate may be important when cells are metabolically active mainly in the nucleolus and at several foci inside the nucleoplasm, (Strzelecka et al., 2010a) and may explain the embryogenesis where it colocalized with coilin in the control cells (Fig. 2, scramble and/or fertility defects observed in coilin-knockout mice (Tucker control). In the knockdown HEK293T cells, NOLC1 localized in et al., 2001; Walker et al., 2009). the nucleolus, but similar to coilin, NOLC1 did not accumulate at TCERG1, previously named CA150 (Suñé et al., 1997), is a foci in the nucleoplasm (Fig. 2; Fig. S2). In overexposed images, we involved in splicing regulation and transcriptional did not detect NOLC1 localized in residual coilin foci (Fig. 2, elongation. TCERG1 binds to splicing components (Goldstrohm overexposed), which further suggests the loss of CB integrity upon et al., 2001; Lin et al., 2004; Sánchez-Álvarez et al., 2006; TCERG1 knockdown. Smith et al., 2004), co-purifies with spliceosomal subcomplexes TCERG1-depleted cells were also analyzed for changes in other (Deckert et al., 2006; Makarov et al., 2002; Neubauer et al., 1998; nuclear structures, such as speckles (SRSF2), gems (SMN) and the Wahl et al., 2009), and affects alternative pre-mRNA splicing of histone locus body (LSM11) (Fig. 3). No effects on gems or the minigene reporters (Cheng et al., 2007; Lin et al., 2004; Montes histone locus body were observed upon TCERG1 depletion et al., 2012a,b; Pearson et al., 2008; Sánchez-Hernández et al., (Fig. 3B,C), demonstrating that TCERG1 is an important factor 2012) and in putative cellular targets identified by whole- for CBs but is not essential for other nuclear structures. Depletion of transcriptome analysis upon TCERG1 knockdown (Muñoz-Cobo TCERG1, however, affected the morphology of nuclear speckles, et al., 2017; Pearson et al., 2008). Consistent with these findings, which appeared smaller and less diffusely distributed (Fig. 3A). TCERG1 is enriched at the interface between speckles (the Consistent with these findings, TCERG1 was previously found at compartments enriched in splicing factors) and nearby transcription the interface between speckles and nearby transcription and sites (Sánchez-Álvarez et al., 2006; Sánchez-Hernández et al., 2016). processing sites (Sánchez-Álvarez et al., 2006). We also In addition to this location, TCERG1 is distributed throughout performed quantitative analysis of the number of histone locus the nucleoplasm, with increased levels in many other granule-like bodies and nuclear gems in control and knockdown cells, and we sites besides speckles, suggesting additional functions for TCERG1. did not observe significant differences among the samples (Fig. S3). Therefore, we undertook a detailed study on the effects of TCERG1 Using the two knockdown HEK293T cell lines, we assessed knockdown on nuclear architecture. In this study, we show that, upon whether transient TCERG1 overexpression could result in knockdown of TCERG1, there are changes in the integrity of CBs, restoration of the phenotype. In fact, overexpression of a limited and this phenotype is associated with altered snRNP assembly, amount of TCERG1 resulted in significant levels of coilin staining thereby suggesting a new role for TCERG1 in CB formation and in each of the two cell lines (Fig. 4A). Quantification analysis of snRNP biogenesis. CBs showed an increase in the number and size of these organelles in the TCERG1-knockdown HEK293T cells upon TCERG1 re- RESULTS expression (Fig. 4B). These results further support a role for Knockdown of TCERG1 affects CBs TCERG1 in the formation of CBs. To investigate whether TCERG1 plays a role in the formation or maintenance of nuclear bodies, TCERG1 was depleted in TCERG1 associates with CBs HEK293T and HAP1 cells using CRISPR-Cas9 gene editing To examine the presence of TCERG1 in CBs, we stained HEK293T technology, and the effect on CBs, speckles, gems, and histone cells with antibodies against TCERG1 and the CB marker coilin. locus body was analyzed by immunofluorescence microscopy. We We observed that TCERG1 is absent at the nucleolar compartment generated two different HEK293T clones (1AC4 and 2AC2) and and distributed throughout the nucleoplasm with an increased signal one HAP1 clone (c012). In control cells treated with a scramble in organized granule-like sites corresponding to the speckles guide RNA with no homology to any gene (scramble control), coilin (Fig. 5A, panel a), as previously shown (Sánchez-Álvarez et al., displayed the characteristic CB localization (Fig. 1A). Strikingly, no 2006). Dual labeling of cells with antibodies directed against

CBs were found in TCERG1-depleted cells. With an extreme TCERG1 and coilin showed that TCERG1 partially overlapped but Journal of Cell Science

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Fig. 1. Knockdown of TCERG1 mislocalizes coilin. (A) Immunofluorescence staining of endogenous TCERG1 (green) and the CB marker coilin (red) in TCERG1-knockdown HEK293T and HAP1 cells. Individual staining and merged images of cells stained with the indicated antibodies are shown. Overexposure of coilin panels to show residual small foci is also shown. DAPI labeling (blue) was used to stain the nuclei. White arrowheads indicate examples of Cajal bodies. Scale bars: 7.5 µm. (B) Quantification analysis (mean±s.e.m.) of CB number and size. For HEK293T cells, the number of nuclei measured was n=37(control) and n=31 (1AC4 and 2AC2); for HAP1 cells, n=46 (control) and n=51 (C012). *P≤0.05, **P≤0.01, ***P≤0.001. (C) Western blotting of samples from knockdown HEK293T and HAP1 cells and HEK293T cells transiently transfected with siRNAs against TCERG1. The antibodies used are listed on the right. (D) The graph shows the quantification of coilin mRNA expression upon TCERG1 depletion using siRNAs. The data are from three independent experiments (mean±s.e.m.). did not coincide with the coilin-containing bodies (Fig. 5A, panels phases by using hydroxyurea (Fig. 5B,C, panel f). We observed an c,d), suggesting that TCERG1 is enriched at the CB periphery. The increased level of colocalization during G1 phase (Fig. 5B,C, spatial relationship between TCERG1 relative to coilin was compare panel d and e) when, presumably, more actively identified by quantitatively scanning specific nuclear regions transcribing snRNA genes are recruited to the CB periphery. containing CBs (Fig. 5A, panel e). To assess whether the level of Interestingly, immunoprecipitation of endogenous TCERG1 colocalization of TCERG1 and coilin changes during the cell cycle, revealed that it associates with coilin (Fig. 5D). Given that the we obtained synchronous populations of cells arrested in G1 and S1 washes of the immunoprecipitation were performed with RIPA Journal of Cell Science

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Fig. 2. Knockdown of TCERG1 affects NOLC1 localization in Cajal bodies. Immunofluorescence of TCERG1-knockdown HEK293T cells stained against NOLC1 (green) and coilin (red). DAPI labeling (blue) was used to stain the nuclei. An overexposure of panels to show residual small coilin foci is also shown. White arrowheads indicate Cajal bodies. Scale bar: 7.5 µm.

buffer (see Materials and Methods), the association of TCERG1 stable ring-like structure of the core snRNPs in the cytoplasm, with coilin is fairly strong. TCERG1 is also associated with CRM1, which is followed by nuclear import and snRNP maturation in CBs SMN and integrator complex subunit 11 (INTS11) (Fig. 5E), which (Matera and Wang, 2014). We have previously shown that TCERG1 are factors involved in CB homeostasis and snRNP biogenesis. interacts with Sm proteins in vitro and in vivo (Sánchez-Álvarez These results suggest that TCERG1 is present at CBs and is et al., 2006). To investigate whether TCERG1 plays important roles associated with these bodies through its interaction with coilin and/ in snRNP formation, we performed immunoprecipitation or other CB components. experiments with anti-Sm (Y12) antibodies using cell lysates from TCERG1-knockdown and scramble control cells, and Defects in snRNP formation upon TCERG1 knockdown quantified the amount of associated RNA by real-time The complex process of snRNP biogenesis requires the assembly of quantitative reverse transcription PCR (RT-qPCR). We observed a seven Sm proteins around the Sm-binding site of snRNAs to form a reduction in the amount of precipitated U1, U2, U4, U5 and U6

Fig. 3. Knockdown of TCERG1 causes no effect on speckles, gems or histone locus bodies. Dual labeling of cells with antibodies directed against TCERG1 (green) and the speckle marker SRSF2 (red) (A), TCERG1 (green) and the gems marker SMN (red) (B), and the histone locus body marker LSM11 (green) and coilin (red) (C) in TCERG1-knockdown HEK293T and HAP1 cells were performed. DAPI labeling (blue) was used to stain the nuclei. White arrowheads indicate examples of nuclear bodies. Scale bars: 7.5 µm. Journal of Cell Science

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Fig. 4. Re-expression of TCERG1 rescues the phenotype in TCERG1-knockdown cells. (A) Individual staining and merged images of cells stained with TCERG1 (green) and coilin (red) antibodies in TCERG1-knockdown HEK293T cells transiently transfected with empty vector or TCERG1 expression vector are shown. White arrowheads indicate examples of Cajal bodies in the TCERG1- overexpressing cells. Scale bars: 7.5 µm. (B) Quantification analysis of CB number and size. The number of nuclei measured was n=86 (scramble control), n=40 (1AC4+empty vector), n =94 (1AC4+TCERG1), n=40 (2AC2+empty vector) and n=5 (2AC2+TCERG1). *P≤0.05, **P≤0.01, ***P≤0.001.

snRNAs in the lysates with knockdown of TCERG1 compared RT-qPCR reactions with primers binding to the 3′-end extension of with those from control lysates (Fig. 6A). Similar results pre-U1 and pre-U2 snRNA. We did not detect an interaction between were obtained with the TCERG1-knockdown HAP1 cells TCERG1 and pre-U1 and pre-U2 RNA (data not shown). TCERG1 (Fig. 6B). To corroborate these results, we performed a similar interacts with mature U2 snRNP (data not shown), which is in experiment after TCERG1 overexpression. When TCERG1 was agreement with the reported association of TCERG1 with U2AF65 overexpressed in HEK293T cells, an increased amount of all the (Sánchez-Álvarez et al., 2006) and SF1 (Goldstrohm et al., 2001) and precipitated snRNAs was observed (Fig. 6C). To rule out the the presence of TCERG1 in the first spliceosome complex (Neubauer possibility of a decreased amount of U-rich snRNAs in the depleted et al., 1998; Zhou et al., 2002). We also investigated the role of samples, we quantified the amount of U1, U2, U4, U5, and U6 TCERG1 in the expression of pre-U1 and pre-U2 snRNAs. We RNAs in HEK293T and HAP1 cells by RT-qPCR. We did not performed RT-qPCR experiments to measure the levels of pre-U1 observe decreased levels of snRNPs in the cells upon TCERG1 and pre-U2 snRNA precursors upon TCERG1 knockdown. We knockdown or increased levels upon TCERG1 overexpression observed similar levels of pre-U1 and pre-U2 snRNA precursors in (Fig. 6D–F). In contrast, TCERG1 depletion caused a reproducible, control and TCERG1-depleted cells (Fig. S4). Taken together, these although modest, increase in snRNA levels (Fig. 6D,E). results did not support a direct transcriptional effect of TCERG1 in Interestingly, no detectable change of the endogenous Sm the expression of these snRNAs. proteins was observed under conditions of TCERG1 gene To further investigate the role of TCERG1 in snRNP production, knockdown or overexpression (Fig. 6G), and a similar amount of we analyzed whether nascent snRNP production was affected upon Sm protein was precipitated with the Y12 antibody from knockdown of TCERG1. We took advantage of a U2OS cell line knockdown and scramble control cells (Fig. 6H). Overall, these stably expressing GFP–SmB under a doxycycline (dox)-inducible results indicate that TCERG1 is involved in the recruitment of Sm promoter that was previously generated to monitor nascent snRNP proteins to spliceosomal snRNAs. biogenesis from the pool of previously assembled snRNPs (Hutten A defect in snRNP assembly upon TCERG1 knockdown would et al., 2014). As expected (Hutten et al., 2014), immunoblotting of provoke a partially assembled U1 and U2 snRNP accumulation in total cell lysates after 20 or 48 h of dox induction revealed high TCERG1-depleted cells. To test this possibility, we used antibodies expression of GFP–SmB with no change in TCERG1 or CDK9 against the 5′-end of the snRNA trimethyl cap structure (TMG) to (control) expression (Fig. 7A), and GFP–SmB localized correctly in perform co-immunoprecipitations. We observed an increase in CBs and nuclear speckles (Fig. 7B). Next, we used specific TMG-capped U1 snRNA, while the levels of TMG-capped U2 antibodies against the TMG cap to precipitate snRNP and GFP– snRNA were similar to control cells upon TCERG1 depletion SmB complexes in control and TCERG1-knockdown cell extracts (Fig. 6I). The results obtained with TMG-capped U1 snRNA are after 18 h of dox induction, followed by SDS-PAGE and consistent with our previous data suggesting a defect in snRNP immunoblotting for GFP–SmB detection. Strikingly, we observed assembly in the absence of TCERG1. a reduced amount of GFP–SmB in the immunoprecipitations from To test whether TCERG1 binds directly to pre-snRNAs, we TCERG1-depleted cells (Fig. 7C). These results suggest that the performed immunoprecipitation experiments with TCERG1-specific production of nascent snRNA particles is inhibited upon TCERG1

antibodies, and the resulting pellets were subsequently analyzed in depletion. Journal of Cell Science

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Fig. 5. Association of TCERG1 with Cajal bodies and coilin. (A) Dual labeling of HEK293T cells with antibodies directed against TCERG1 (green) and coilin (red) was performed. Individual staining (a, b), merge (c), and colocalization (d) images of cells stained with the indicated antibodies are shown. A merge of signals is defined as the overlap of two of the emission signals due to their close proximity (in yellow). The colocalization of signals is defined by the presence of two signals in the same image pixel with an intensity profile above that of a given fluorescence background. Line scans showing local intensity distributions of TCERG1 (green) and coilin (red) are also shown (e). Scale bar: 5 µm. (B,C) Colocalization analysis of TCERG1 (green) and coilin (red) in G1 (B) and S (C) phase HEK293T cells. Individual staining (a, b), merge (c), and colocalization (d) images of cells stained with anti-TCERG1 and anti-coilin antibodies are shown. Line scans showing local intensity distributions of TCERG1 (green) and coilin (red) are also shown (e). Flow cytometry analysis of the cell cycle profile of cells arrested in G1 (B, panel f) and S (C, panel f) using hydroxyurea is also shown. Scale bars: 7.5 µm. (D,E) TCERG1 associates with factors involved in CB homeostasis. (D) Whole-cell fractions of HEK293T cells were subjected to immunoprecipitation (IP) with TCERG1- specific antibodies followed by SDS-PAGE and western blotting analysis to detect coilin. The amount of total input was 10% of the amount of total protein used in each pull down, and the amount of total output (IP) was 30% of the pull down. (E) HEK293T cells were transiently transfected with a plasmid encoding T7-tagged TCERG1 or an empty vector as a negative control. Whole-cell extract fractions were prepared and directly analyzed by western blotting or subjected to immunoprecipitation (IP) with T7-specific antibodies followed by SDS-PAGE and western blotting using antibodies to detect coilin, SMN, CRM1 and integrator (INTS11). White spaces in the blots indicate removal of empty or loading control lanes. Molecular masses in kDa are indicated to the left.

DISCUSSION knockdown in combination with immunoprecipitation, we detected Our data indicate that TCERG1 plays an important role in the a striking effect of reduced TCERG1 expression on the association assembly of Sm–snRNA complexes during the biogenesis of of Sm proteins with snRNAs to form the minimal core snRNP snRNPs and the formation of CBs. By performing TCERG1 (Fig. 6A,B). A lower Sm core assembly was also observed in vivo Journal of Cell Science

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Fig. 6. TCERG1 modulates the recruitment of Sm proteins to spliceosomal snRNAs. (A,B) Knockdown of TCERG1 reduces the recruitment of Sm to snRNAs. Total cell lysates from TCERG1-knockdown HEK293T (A) or HAP1 (B) cells were immunoprecipitated with anti-Y12 antibodies. The bound RNAs were extracted, and we subsequently performed RT-qPCR assay to detect the relative fold changes of precipitated U1, U2, U4, U5 and U6 snRNAs. Control: scramble siRNA-transfected cells. (C) The same experimental procedures described in A and B were performed in HEK293T cells transiently transfected with a plasmid encoding TCERG1 or an empty vector as a negative control. Data in A–C are from three independent experiments (mean±s.e.m.). (D–F) RNAs in total lysate samples from TCERG1-knockdown HEK293T (D) and HAP1 (E) cells and from TCERG1-overexpressing HEK293T cells (F) were isolated. We then performed RT-qPCR assay to detect the relative fold changes of U1, U2, U5, and U6 snRNAs. The data are from four independent experiments (means±s.e.m.). *P≤0.05. (G) Whole-cell fractions of the indicated cells were subjected to SDS-PAGE and western blotting analysis with anti-TCERG1, anti-Sm (Y12) and anti- CDK9 antibodies. OE, overexpression. (H) Total lysates of different samples were immunoprecipitated with anti-Sm (Y12) or anti-IgG as a negative control. The co-precipitated proteins were subjected to SDS-PAGE and blotted with anti-Y12 antibody. The arrowhead marks the position of Y12, the asterisk (*) marks a cross-reacting band, and the double asterisk (**) marks a cross-reaction with the antibody chains. Molecular masses in kDa are indicated to the left. A longer exposure of the same gel is shown at the bottom of the figure to justify the presence of the cross-reacting band (*) in the input lanes. (I) Total lysates of control and TCERG1-knockdown cells were immunoprecipitated (IP) with anti-TMG cap antibody. The bound RNAs were extracted. We subsequently performed RT-qPCR to detect the relative fold changes of precipitated U1 and U2 snRNAs. The data are from three independent experiments (means±s.e.m.). using a nascent GFP–SmB fusion protein (Fig. 7C). Given that the Álvarez et al., 2006) and coilin (Fig. 5), published data implicating endogenous levels of U snRNAs were not decreased in these TCERG1 in the functional regulation of the splicing process (Cheng conditions (Fig. 6D), the effect of TCERG1 on Sm–snRNP et al., 2007; Lin et al., 2004; Montes et al., 2012a,b; Muñoz-Cobo formation is unlinked to snRNA transcription. The increased et al., 2017; Pearson et al., 2008; Sánchez-Hernández et al., 2012), snRNA levels observed upon TCERG1 knockdown are surprising, and the implication of TCERG1 in snRNP biogenesis described in given that snRNP assembly is inhibited, and the increase could this study has led us to hypothesize that TCERG1 takes part in Sm– reflect cellular responses to perturbations in the formation of mature snRNP formation in the cytoplasm and then facilitates the splicing snRNP complexes. These snRNAs might be stabilized by other process in the nucleus. Our results reveal the exciting possibility that proteins, or their degradation pathways might be affected. More the effects of TCERG1 on splicing could be due, at least in part, to work is clearly necessary to elucidate the reasons for the snRNA modulating Sm–snRNP formation. increase upon depletion of TCERG1. During the stepwise snRNP Our data also indicate that TCERG1 participates in the formation biogenesis pathway, snRNAs move from the nucleus to the or integrity of CBs. CBs are conserved nuclear bodies involved in cytoplasm. In the cytoplasm, the Sm assembly of the snRNPs the final biogenesis of snRNPs, which are essential factors for pre- occurs followed by their import to different nuclear compartments, mRNA splicing. Despite being discovered in vertebrates more than such as speckles and CBs, to participate in the process of pre-mRNA 100 years ago, studies on the molecular requirements for the splicing. Although TCERG1 has been identified as a nuclear formation and stability of CBs began two decades ago and these protein, it may shuttle between the nucleus and cytoplasm, as details have not been thoroughly elucidated to date. Interestingly, suggested in a recent report showing the presence of TCERG1 in the knockdown of TCERG1 disrupts CBs without affecting other cytoplasm of neuronal cells (Muñoz-Cobo et al., 2017). The nuclear structures (Figs 1–3). The effect of TCERG1 depletion observed interaction of TCERG1 with Sm proteins (Sánchez- mimics the effect of the loss of coilin, which produces defects in CB Journal of Cell Science

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Fig. 7. Production of nascent snRNP is affected upon TCERG1 knockdown. (A) GFP–snRNP expression in the U2OS cell line. Total cell lysates from GFP–SmB-expressing U2OS cells without (−) or 20 and 48 h after (+) doxycycline (dox) induction were analyzed for GFP–SmB expression by western blotting using anti-GFP and the indicated antibodies. Molecular masses in kDa are indicated to the left. (B) Dual labeling of U2OS GFP-SmB cells for GFP (green) and with antibodies directed against coilin (red) and SRSF2 (red), as markers for CBs and speckles, respectively, was performed. Individual staining and merged images of cells stained with the indicated antibodies are shown. DAPI labeling (blue) was used to stain the nuclei. White arrowheads indicate Cajal bodies. Scale bars: 7.5 µm. (C) Formation of nascent snRNPs was analyzed by TMG immunoprecipitation in GFP–SmB-expressing U2OS cells treated with siControl or siTCERG1. GFP–SmB expression was analyzed by immunoblotting using GFP-specific antibodies. The asterisk (*) marks a nonspecific band, and the double asterisk (**) marks a cross-reaction with the antibody chains. Molecular masses in kDa are indicated to the left. formation in many organisms (Collier et al., 2006; Liu et al., 2009; the Sm proteins but not naked snRNAs restore the formation of CBs Strzelecka et al., 2010b; Tucker et al., 2001), and the loss of many after their depletion and that the Sm ring is sufficient to target other proteins involved in snRNP maturation, such as SMN (Girard snRNAs to CBs (Roithová et al., 2018) is in keeping with this et al., 2006; Lemm et al., 2006), TGS1 (Lemm et al., 2006), PHAX model. Future experiments will determine how the roles of (Lemm et al., 2006), INTS4 (Takata et al., 2012), WRAP53 TCERG1 in CB and snRNP formation described in this study are (Mahmoudi et al., 2010) and USPL1 (Hutten et al., 2014), that also directly linked to the functional roles of this protein in transcription disrupt CBs. and alternative splicing. What is the mechanism by which TCERG1 affects CB integrity? TCERG1 is present at the periphery of CBs (Fig. 5A), suggesting MATERIALS AND METHODS that TCERG1 is neither a constitutive component of CBs nor assists Plasmids and antibodies coilin as a scaffold protein in the formation of CBs. One possibility The mammalian expression vectors pEFBOST7-TCERG1 and pEFBOS/ is that TCERG1 affects CB integrity by controlling the expression of GFP/T7-TCERG1[1-1098] have been described previously (Sánchez- an integral component of this . However, we did not find Álvarez et al., 2006; Sánchez-Hernández et al., 2016; Suñé and Garcia- any relevant known protein component of CBs (Machyna et al., Blanco, 1999). 2013) in which transcription or alternative splicing is affected by For immunoblotting, the following primary antibodies were used at the TCERG1 depletion using data from available transcriptome studies indicated dilution: anti-coilin (sc-55594; Santa Cruz Biotechnology, Dallas, (Muñoz-Cobo et al., 2017; Pearson et al., 2008). Although TX) at 1:1000; anti-CDK9 (sc-484; Santa Cruz Biotechnology) at 1:2000; TCERG1 interacts with SMN (Cheng et al., 2007) (Fig. 5E) and anti-Sm (Y12) (MS-450; Thermo Fisher Scientific, Waltham, MA) at SMN affects Sm core assembly displaying defects in CB formation 1:1000; anti-SMN (sc-32313; Santa Cruz Biotechnology) at 1:500; anti- (Raimer et al., 2017 and references therein), our results show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-365062; Santa Cruz Biotechnology) at 1:2000; and anti-CRM1 and anti-INTS11 at 1:1000 SMN localization to gems remained unaffected upon TCERG1 (generously provided by Edouard Bertrand, IGMM-CNRS, Montpellier, knockdown (Fig. 3), thereby making SMN unlikely to be the factor France). The TCERG1 antibodies used in the western blot analysis in this involved in destabilizing these nuclear bodies through altering study were generated in guinea pigs using two truncated glutathione S TCERG1 expression levels. The consequences of the interaction of transferase (GST) fusion TCERG1 proteins spanning amino acids 234 to TCERG1 with other CB factors (Fig. 5E) in the formation of these 662 and 631 to 1098, and were used at a dilution of 1:10,000. For nuclear structures have not been determined. The formation of CBs immunofluorescence studies, we used the following antibodies: anti- also depends on the presence of snRNPs (Lemm et al., 2006; TCERG1 (Suñé et al., 1997) at 1:2000; anti-coilin at 1:500; anti-T7 (A190- Sleeman et al., 2001). Furthermore, immature snRNPs induce the 116A; Bethyl Laboratories, Montgomery, TX) at 1:1000; anti-NOLC1 formation of CBs in cells lacking these nuclear structures, while (ab106324; Abcam, Cambridge, UK) at 1:500; anti-SRSF2 (S4045; Sigma, mature particles fail to generate CBs (Novotný et al., 2015; Roithová St Louis, MO) at 1:4000; anti-Sm at 1:50, anti-SMN at 1:200 and anti- LSM11 (generously provided by Joseph G. Gall, Carnegie Institution for et al., 2018). A self-assembly model via coilin, in which different Science, Baltimore, MD) at 1:200. For western blot analysis, primary RNA species nucleate residual or sub-CBs that fuse to assemble a antibodies were detected using HRP-conjugated secondary antibodies to mature CB, has been recently proposed (Machyna et al., 2013). rabbit-IgG and mouse-IgG (PerkinElmer Life Sciences; Waltham, TCERG1 may disrupt CBs by interfering with the formation of the Massachusetts) at a dilution of 1:5000. For immunofluorescence, we used snRNP-dependent sub-CB by inhibiting core Sm–snRNP Alexa Fluor 488-conjugated goat anti-rabbit-IgG, Alexa Fluor 550- formation. The recent observation that core snRNPs containing conjugated goat anti-rabbit-IgG, and Alexa Fluor 647-conjugated goat Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs232728. doi:10.1242/jcs.232728 anti-mouse-IgG antibodies from Molecular Probes (Eugene, Oregon) at a fluoride (PMSF), and protease inhibitor mixture (Complete Roche)], dilution of 1:500. RIPA buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM dithiothreitol, Cell culture and transfection assays 1 mM PMSF, and protease inhibitor mixture) for 30 min at 4°C. The cell HEK293T cells were originally obtained from the American Type Culture extracts were centrifuged at maximum speed (16,100 g)for5min.The Collection (ATCC, Manassas, VA) and were grown and maintained as proteins were separated by SDS-PAGE, transferred to a nitrocellulose described previously (Sánchez-Álvarez et al., 2010). Transfection assays were membrane (Amersham Biosciences; Little Chalfont, UK) and then carried out using protocols described previously (Sánchez-Álvarez et al., 2006, incubated with specific antibodies. After washing, the membrane was 2010). Cells were transfected using calcium phosphate or Lipofectamine 2000 incubated with peroxidase-conjugated secondary antibodies, and the reagent (Invitrogen, Carlsbad, CA) according to the protocols of the bound antibodies were detected by enhanced chemiluminescence manufacturer. Clonal U2OS GFP–SmB-expressing cells were generously (PerkinElmer Life Science). provided by Angus I. Lamond (University of Dundee, UK) and were grown and maintained in Dulbecco’s modified Eagle medium (Invitrogen) Immunoprecipitation experiments supplemented with 10% fetal bovine serum, penicillin–streptomycin 100 U and 100 μg/ml, respectively, 150 mg/ml hygromycin B, and 15 mg/ml Cells were harvested into 500 ml of RIPA buffer for 30 min at 4°C, and the blasticidin-HCl (Hutten et al., 2014). Expression of GFP–SmB was induced cell extracts were centrifuged at 16,100 g for 5 min at 4°C. 10% fractions of with 10 ng/ml doxycycline (Sigma) for 18 h. The source of SH-SY5Y cells whole cell extract (WCE) were boiled in SDS-PAGE loading buffer and saved μ was reported elsewhere (Muñoz-Cobo et al., 2017) and were grown and for immunoblotting analysis. The supernatants were incubated with 3 lof μ μ maintained as described previously (Muñoz-Cobo et al., 2017). The scramble, control mouse, rabbit IgG or Y12 (0.6 g), anti-TMG (0.75 g; MABE302, μ 1AC4 and 2AC2 cell lines were obtained using a TCERG1 human gene Millipore) or anti-TCERG1 antibodies (3 l) overnight at 4°C and then knockdown kit in HEK293T cells according to the manufacturer’s instructions incubated with Protein A/G agarose beads (Millipore, Burlington, MA) with (Origene, Rockville, MD). Cells were cultured in Dulbecco’s modified Eagle end-over-end rotation for 2 h at 4°C. The anti-TMG antibodies (K121) have medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin– high affinity for trimethylguanosine and cross-react weakly with 7- streptomycin (100 U and 100 μg/ml), and 1.5 μg/ml puromycin. TCERG1- methylguanosine at very high concentration (Krainer, 1988). Because the knockdown HAP1 cells (Horizon Discovery, Waterbeach, Cambridge, UK) anti-TMG antibodies preferentially bind to the trimethylguanosine, they have were maintained in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, been routinely used to detect specifically the trimethyl cap versus the m7G Thermo Fisher Scientific) supplemented with 10% FBS (Gibco) and cap. After six washes with RIPA buffer, the proteins bound to the antibody penicillin-streptomycin 100 U and 100 μg/ml, respectively. All cell lines resin were eluted by boiling the samples with SDS-PAGE loading buffer were tested regularly for contamination. (0.2% Bromophenol Blue, 100 mM DTT, SDS 4%, 20% glycerol and Tris- For the experiments with cells synchronized for the cell cycle, cells were HCl 0.1 M, pH 6.8), separated on 10% SDS-PAGE gels, and analyzed by seeded on sterile glass coverslips and synchronized at G1 phase by western blotting. RNA immunoprecipitation was performed as previously incubation with 0.5 mM hydroxyurea (Thermo Fisher Scientific) for 18 h. described (Niranjanakumari et al., 2002) with minor modifications. Briefly, For synchronization at S phase, cells were then incubated in normal DMEM cells were fixed with 1% formaldehyde for 10 min at room temperature with for 3 additional hours before being fixed. Synchronized cells were also slow mixing; cross-linking was arrested by adding glycine at 0.125 M for analyzed using a FACSAria III cell sorter flow cytometer (Becton– 5 min at room temperature. Cells were lysed in RIPA buffer and sonicated 3 Dickinson). Data were evaluated using FlowJo cell analysis software. times for 20 s on ice. Supernatants were precleared with protein A/G-agarose For RNA interference (siRNA) knockdown experiments, HEK293T and fast-flow slurry (Millipore) and tRNA at 100 µg/µl with end-over-end rotation U2OS cells were seeded to ∼50% confluence in 35 mm plates. The cells for 1 h at 4°C. Beads were removed by centrifugation, and the supernatant was were transfected using the Lipofectamine 2000 reagent (Invitrogen) for incubated with nonspecific mouse IgG and specific antibodies overnight. The – – HEK293T and Lipofectamine RNAiMaxx (Invitrogen) for U2OS cells RNA antibody complexes were collected with protein A/G agarose beads according to the manufacturer’s protocol with 60 nM (final concentration) for 2 h at 4°C. Subsequently, the beads were collected and washed six times of either of the following small interfering RNA (siRNA) duplexes: siEGFP, with RIPA buffer and resuspended in a buffer containing 100 µl of 50 mM 5′-CUACAACAGCCACAACGCU-3′; siControl, 5′-CAGUCGCGUUU- Tris-HCl pH 7.0, 5 mM EDTA, 10 mM dithiothreitol and 1% SDS with a GCGACUGG-3′; siTCERG1, 5′-GGAGUUGCACAAGAUAGUU-3′. final incubation at 70°C for 45 min.

Immunofluorescence analysis and treatments RNA extraction and RT-PCR analysis Immunofluorescence staining in HEK293T and HAP1 cells was performed Total RNA was extracted from transfected cells with peqGOLD TriFast as previously described (Sánchez-Hernández et al., 2017). SH-SY5Y cells (peQlab, Fareham, UK) according to the manufacturer’s protocol. were grown on coverslips and fixed with 4% paraformaldehyde and 4% Approximately 1 μg of RNA was digested with 10 units of RNase-free sucrose in PBS (pH 7.4) for 20 min at 37°C and permeabilized with 90% DNase (Ambion, Foster City, CA), and RNA was reverse transcribed using chilled methanol for 5 min. The cells were blocked in PBS containing 10% the qScript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD) BSA and 0.5% Triton X-100 for 1 h at room temperature. The cells were following the manufacturer’s protocol. Quantification of endogenous incubated with primary antibody in PBS containing 10% BSA for 2 h at transcripts by real-time PCR was performed using the iQ SYBR Green room temperature (humidity chamber). The cells were subsequently washed Supermix (Bio-Rad, Hercules, CA) and the iCycler thermal cycler station three times with 0.1% BSA in PBS. This step was followed by labeling with (Bio-Rad) with the following oligonucleotides: Coilin-Fw, 5′-CTTGAG- secondary antibodies for 2 h at room temperature (humidity chamber). The AGAACCTGGGAAATTTG-3′ and Coilin-Rv, 5′-GTCTTGGGTCAAT- cells were subsequently washed three times with 0.1% BSA in PBS and CAACTCTTTCC-3′; U1-Fw, 5′-GATACCATGATCACGAAGGTGGTT- three times with PBS. Coverslips were mounted onto glass slides using the 3′ and U1-Rv, 5′-CACAAATTATGCAGTCGAGTTTCC-3′; U2-Fw, ProLong Gold Antifade reagent with DAPI (Life Technologies, Carlsbad, 5′-ATCGCTTCTCGGCCTTTTGG-3′ and U2-Rv, 5′-GGTGCACCGTTC- California). Images were acquired using a Leica SP5 spectral laser confocal CTGGAGG-3′;U4-Fw,5′-GCGCGATTATTGCTAATTGAAA-3′ and U4- microscope and processed using the LAS AF software v2.3.6. Rv, 5′-AAAAATTGCCAATGCCGACTA-3′;U5-Fw,5′-GGTTTCTCTTC- Quantification was performed using ImageJ software. The images were AGATCGCATAAATC-3′ and U5-Rv, 5′-CTCAAAAAATTGGGTTAAGA- digitally processed for presentation using the Adobe Photoshop CS3 CTCAGA-3′;U6-Fw,5′-GCTTCGGCAGCACATATACTAAAAT-3′ and extended v10.0 software. U6-Rv, 5′-ACGAATTTGCGTGTCATCCTT-3′;preU1-Fw,5′-ACTGCGTT- CGCGCTTTCCC-3′;preU1-Rv,5′-GCAGGCGACATGTTACTTCC-3′; Western blot analysis preU2-Fw, 5′-AACATAGGTACACGTGTGCCACGG-3′;preU2-Rv,5′-AC- Cells were lysed in T7 buffer [20 mM HEPES pH 7.9, 150 mM NaCl, 5 mM AAATAGCCAACGCATGCGGGGC-3′. GAPDH was used as an internal

EDTA, 1% NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl control gene and was amplified with the oligonucleotides GAPDH-Fw Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs232728. doi:10.1242/jcs.232728

(5′-ATGGGGAAGGTGAAGGTCG-3′) and GAPDH-Rv (5′-GGG TCATT- Goldstrohm, A. C., Albrecht, T. R., Suñé, C., Bedford, M. T. and Garcia-Blanco, GATGGCAACAATATC-3′). Statistical analysis was performed using Microsoft M. A. (2001). The transcription elongation factor CA150 interacts with RNA Mol. Cell. Biol. Excel 2010 and GraphPad Prism 5.0 software. polymerase II and the pre-mRNA splicing factor SF1. 21, 7617-7628. doi:10.1128/MCB.21.22.7617-7628.2001 Hu, D., Smith, E. R., Garruss, A. S., Mohaghegh, N., Varberg, J. M., Lin, C., Statistical analysis Jackson, J., Gao, X., Saraf, A., Florens, L. et al. (2013). The little elongation All experiments were repeated at least three times, and statistical analysis complex functions at initiation and elongation phases of snRNA gene was performed using Prism 5.0 software (GraphPad). Two-tailed Student’s transcription. Mol. Cell 51, 493-505. doi:10.1016/j.molcel.2013.07.003 tests (unpaired t-tests) were used to compare the samples and their respective Hutten, S., Chachami, G., Winter, U., Melchior, F. and Lamond, A. I. (2014). A P P≤ P≤ role for the Cajal-body-associated SUMO isopeptidase USPL1 in snRNA controls. The -values are represented by asterisks (* 0.05, ** 0.01 and J. Cell Sci. P≤ transcription mediated by RNA polymerase II. 127, 1065-1078. *** 0.001). The absence of an asterisk indicates that the change relative to doi:10.1242/jcs.141788 the control is not statistically significant. Isaac, C., Yang, Y. and Meier, U. T. (1998). Nopp140 functions as a molecular link between the nucleolus and the coiled bodies. J. Cell Biol. 142, 319-329. doi:10. Acknowledgements 1083/jcb.142.2.319 We thank Joseph Gall for the anti-LSM11 antibody, Edouard Bertrand for the anti- Klingauf, M., Stanĕk, D. and Neugebauer, K. M. (2006). Enhancement of U4/U6 CRM1 and anti-INTS11 antibodies, and Angus I. Lamond for the U2OS GFP-SmB small nuclear ribonucleoprotein particle association in Cajal bodies predicted by Mol. Biol. Cell cells. We are grateful to David Staněk for experimental conversations and advice. mathematical modeling. 17, 4972-4981. doi:10.1091/mbc.e06-06- The technical assistance of Laura Montosa during the confocal microscopy studies 0513 is deeply appreciated. Krainer, A. R. (1988). Pre-mRNA splicing by complementation with purified human U1, U2, U4/U6 and U5 snRNPs. Nucleic Acids Res. 16, 9415-9429. doi:10.1093/ nar/16.20.9415 Competing interests Lardelli, R. M., Schaffer, A. E., Eggens, V. R. C., Zaki, M. S., Grainger, S., Sathe, The authors declare no competing or financial interests. S., Van Nostrand, E. L., Schlachetzki, Z., Rosti, B., Akizu, N. et al. (2017). Biallelic mutations in the 3′ exonuclease TOE1 cause pontocerebellar hypoplasia Author contributions and uncover a role in snRNA processing. Nat. Genet. 49, 457-464. doi:10.1038/ Conceptualization: C.M.-C., S.P.-S., C.H.-M., C.S.; Methodology: C.M.-C., S.P.-S.; ng.3762 Validation: C.M.-C.; Formal analysis: C.M.-C.; Investigation: C.M.-C., N.S.-H.; Lemm, I., Girard, C., Kuhn, A. N., Watkins, N. J., Schneider, M., Bordonné,R. Resources: S.P.-S., C.H.-M., C.S.; Writing - original draft: C.M.-C., C.S.; Writing - and Lührmann, R. (2006). Ongoing U snRNP biogenesis is required for the review & editing: C.H.-M., C.S.; Visualization: C.M.-C., S.P.-S., C.S.; Supervision: integrity of Cajal bodies. Mol. Biol. Cell 17, 3221-3231. doi:10.1091/mbc.e06-03- C.H.-M., C.S.; Project administration: C.S.; Funding acquisition: C.H.-M., C.S. 0247 Li, Y., Fong, K.-W., Tang, M., Han, X., Gong, Z., Ma, W., Hebert, M., Songyang, Z. Funding and Chen, J. (2014). Fam118B, a newly identified component of Cajal bodies, is required for Cajal body formation, snRNP biogenesis and cell viability. J. Cell Sci. This work was supported by grants from the Spanish Ministry of Economy and 127, 2029-2039. doi:10.1242/jcs.143453 Competitiveness (Ministerio de Economıá y Competitividad; grant number Lin, K.-T., Lu, R.-M. and Tarn, W.-Y. (2004). The WW domain-containing proteins BFU2017-89179-R to C.S. and grant number BFU2016-79699-P to C.H.M.) and the interact with the early spliceosome and participate in pre-mRNA splicing in vivo. Andalusian Government (Excellence Project BIO-2515/2012) to C.S. Support from Mol. Cell. Biol. 24, 9176-9185. doi:10.1128/MCB.24.20.9176-9185.2004 the European Region Development Fund [ERDF (FEDER)] is also acknowledged. Liu, J.-L., Wu, Z., Nizami, Z., Deryusheva, S., Rajendra, T. K., Beumer, K. J., Gao, H., Matera, A. G., Carroll, D. and Gall, J. G. (2009). Coilin is essential for Supplementary information Cajal body organization in Drosophila melanogaster. Mol. Biol. 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