RBM 14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity
2014; Springer Nature; Volume: 34; Issue: 1 Linguagem: Inglês
10.15252/embj.201488979
ISSN1460-2075
AutoresGen Shiratsuchi, Katsuyoshi Takaoka, Tomoko Ashikawa, Hiroshi Hamada, Daiju Kitagawa,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle10 November 2014Open Access RBM14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity Gen Shiratsuchi Gen Shiratsuchi Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Katsuyoshi Takaoka Katsuyoshi Takaoka Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Tomoko Ashikawa Tomoko Ashikawa Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Hiroshi Hamada Hiroshi Hamada Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Daiju Kitagawa Corresponding Author Daiju Kitagawa Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Gen Shiratsuchi Gen Shiratsuchi Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Katsuyoshi Takaoka Katsuyoshi Takaoka Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Tomoko Ashikawa Tomoko Ashikawa Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Hiroshi Hamada Hiroshi Hamada Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Daiju Kitagawa Corresponding Author Daiju Kitagawa Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan Search for more papers by this author Author Information Gen Shiratsuchi1, Katsuyoshi Takaoka2, Tomoko Ashikawa1, Hiroshi Hamada2 and Daiju Kitagawa 1 1Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan 2Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan *Corresponding author. Tel: +81 55 981 5828; E-mail: [email protected] The EMBO Journal (2015)34:97-114https://doi.org/10.15252/embj.201488979 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Formation of a new centriole adjacent to a pre-existing centriole occurs only once per cell cycle. Despite being crucial for genome integrity, the mechanisms controlling centriole biogenesis remain elusive. Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes. Depletion of RBM14 in human cells induces ectopic formation of centriolar protein complexes through function of the STIL/CPAP complex. Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity. Moreover, we find that, upon RBM14 depletion, a part of the ectopic centriolar protein complexes in turn assemble into structures more akin to centrioles, presumably by incorporating HsSAS-6, a cartwheel component, and cause multipolar spindle formation. We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre-existing centrioles. This study sheds light on the possibility that ectopic formation of aberrant structures related to centrioles may contribute to genome instability and tumorigenesis. Synopsis New centrioles form by a highly regulated and ordered assembly pathway, starting from a distinctive "cartwheel" structure. Identification and characterization of the novel centriole biogenesis regulator RBM14 uncovers a non-canonical, cartwheel-independent pathway that allows formation of procentriole-like structures even in the presence of pre-existing centrioles. RBM14 is an interactor of the human centriole assembly factor STIL. RBM14 interferes with complex formation of STIL and CPAP during centriole assembly. RBM14 knock-down leads to ectopic formation of functional centriolar protein complexes in cells. De novo assembly of centriolar protein complexes occurs without a cartwheel structure. RBM14 depletion affects spindle assembly and chromosome segregation during mitosis. Introduction Centrosome duplication is tightly orchestrated with cell cycle progression to yield the correct number of centrosomes, which ensures robust formation of a mitotic bipolar spindle and faithful chromosome segregation (Nigg & Raff, 2009). The centrosome consists of a pair of centrioles surrounded by an electron-dense pericentriolar material (PCM). Formation of a new centriole adjacent to each pre-existing centriole is fundamental for centrosome duplication. Centriole formation starts with the assembly of a cartwheel structure onto which microtubules are then added (Brito et al, 2012; Gönczy, 2012). Besides this canonical pathway, the de novo formation of centrioles can be induced in the case of natural/physical loss of pre-existing centrioles (Marshall et al, 2001; Khodjakov et al, 2002; La Terra et al, 2005), in unfertilized Drosophila eggs overexpressing core centriole duplication factors (Peel et al, 2007; Rodrigues-Martins et al, 2007; Dzhindzhev et al, 2010; Stevens et al, 2010a), during early development in the mouse embryo (Courtois & Hiiragi, 2012) or in multicilia formation of terminally differentiated cells (Nigg & Raff, 2009). Although it is believed that the existence of pre-existing centrioles normally suppresses the de novo assembly in proliferating cells, exactly how this suppression is achieved remains unknown. The SAS-6 family of proteins have been recently identified as crucial components of the cartwheel that is essential for centriole formation (Kilburn et al, 2007; Nakazawa et al, 2007; van Breugel et al, 2011; Kitagawa et al, 2011b). Moreover, human SAS-6 (HsSAS-6) overexpression induces formation of multiple procentrioles around a parental centriole, presumably through formation of multiple cartwheels (Strnad et al, 2007). Similar phenotypes have also been observed for overexpression of STIL (SCL/TAL1 interrupting locus) (Kitagawa et al, 2011a; Tang et al, 2011; Arquint et al, 2012; Vulprecht et al, 2012) and polo-like kinase 4 (Plk4) (Kleylein-Sohn et al, 2007), implying that there is a tight relationship between these critical components of the centriole assembly. In fact, in human cells, Plk4 acts upstream of HsSAS-6, regulating the presence of centriolar HsSAS-6 (Kleylein-Sohn et al, 2007; Strnad et al, 2007). In line with the fact that both HsSAS-6 and STIL localize to the proximal end of procentrioles (Kleylein-Sohn et al, 2007; Strnad et al, 2007; Tang et al, 2011; Arquint et al, 2012; Vulprecht et al, 2012), HsSAS-6 and STIL seem to be dependent on one another for their presence at procentrioles (Tang et al, 2011; Arquint et al, 2012; Vulprecht et al, 2012). HsSAS-6 and STIL are needed in turn for CPAP loading (Kohlmaier et al, 2009; Tang et al, 2009, 2011), which presumably promotes subsequent addition of centriolar microtubules and the elongation of procentrioles (Kohlmaier et al, 2009; Schmidt et al, 2009; Tang et al, 2009). Consistently, the induction of procentriole amplification by Plk4 overexpression is mediated by HsSAS-6 and STIL (Kleylein-Sohn et al, 2007; Tang et al, 2011; Arquint et al, 2012; Vulprecht et al, 2012). Given that the function of their relatives in centriole formation is evolutionarily conserved (Dammermann et al, 2004; Delattre et al, 2004; Leidel et al, 2005; Kilburn et al, 2007; Nakazawa et al, 2007; Rodrigues-Martins et al, 2007; Yabe et al, 2007; Culver et al, 2009; Stevens et al, 2010a) and that their relationship in other systems seems to be, at least in part, conserved (Delattre et al, 2006; Pelletier et al, 2006; Kitagawa et al, 2009; Stevens et al, 2010b), these three proteins are critical for the onset of centriole assembly. To conduct faithful centriole duplication, the function and expression levels of the critical components must be strictly regulated across the cell cycle. In this study, we identify RNA-Binding Motif protein 14 (RBM14) as a STIL-interacting protein that interferes with a complex formation of STIL and CPAP. In addition, our data establish that RBM14 suppresses the formation of aberrant structures related to centrioles in the cytoplasm and thus preserves mitotic spindle integrity. Results Depletion of RBM14 induces ectopic formation of centriolar protein complexes We set out to investigate the mechanisms limiting the number of newly formed centrioles. We reasoned that such regulators could physically interact with and directly control critical centriole proteins such as HsSAS-6 and STIL. Accordingly, we employed the mass spectrometry-based proteomic analysis to identify candidate proteins interacting with HsSAS-6 or STIL. Small-scale RNAi screening was subsequently conducted to address their function in centriole formation in human culture cells. This screening led to the identification of RNA-Binding Motif protein 14 (RBM14, also known as COAA, transcription co-activator, or PSP2, paraspeckle protein 2) (Iwasaki et al, 2001; Fox et al, 2002), a potential tumor suppressor conserved within vertebrates (Kang et al, 2008), as a STIL-interacting protein (Supplementary Table S1). We analyzed mitotic cells, which normally have four centrioles marked with centrin signals and assemble a bipolar spindle (Fig 1A). Intriguingly, we discovered that siRNA-mediated depletion of RBM14 resulted in a significant increase of centrin foci in human U2OS (33 ± 2% compared with 7 ± 2% in control cells, P < 0.01, n = 90 in triplicate; Fig 1A and Supplementary Fig S1A), HeLa cells (19 ± 1% compared with 1 ± 1% in control cells, P < 0.01, n = 90 in triplicate; Supplementary Fig S1B) and RPE1 cells (17 ± 4% compared with 3 ± 2% in control cells, P < 0.05, n = 90 in triplicate; Supplementary Fig S1C). We also found a similar phenotype in interphase cells (Supplementary Fig S1D). The amplification of centrin foci was detectable within 24 h after siRNA transfection in U2OS cells without a significant cell cycle arrest or cytokinesis defect (Supplementary Fig S1E and F). It has been previously reported that RBM14 acts as a component of the nuclear paraspeckle and functions in RNA processing and transcription (Auboeuf et al, 2004). The nuclear paraspeckle is a small intranuclear structure that is known to contain RNA and several ribonucleoproteins. We therefore assessed the possibility that RBM14 depletion somehow affects centriole formation through transcriptional regulation. However, mis-localization of RBM14 from the paraspeckle caused by depletion of p54nrb/NONO, a protein essential for paraspeckle organization (Sasaki et al, 2009), did not induce amplification of centriole foci (Supplementary Fig S1H). In addition, expression levels of STIL and other centriole proteins were unaffected by RBM14 depletion (Supplementary Figs S1A, S4A, S5A, and S9B). Furthermore, incubation with 0.5 mM cycloheximide, a translation inhibitor, did not suppress excess formation of centrin foci by RBM14 depletion (Supplementary Fig S1G). Based on these observations, we conclude that the amplification of centrin foci by RBM14 depletion likely stems from reduction of unknown function of RBM14 rather than its known function as a component of the nuclear paraspeckle. Figure 1. Depletion of RBM14 induces ectopic formation of centriolar protein complexes A–E. Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase. Download figure Download PowerPoint Next, to distinguish whether the amplification of centrin foci is because of multiple rounds of centriole duplication or concurrent formation of multiple procentrioles and/or procentriole-like premature structures, we monitored the number of C-Nap1 or Cep164-labeled parental centrioles (Fry et al, 1998; Graser et al, 2007) and Centrobin, a procentriole marker (Zou et al, 2005), foci in mitotic RBM14-depleted cells. We found that 31 ± 4% of the cells harbored an increased number of Centrobin foci (P < 0.05, n = 90 in triplicate; Fig 1B), whereas the number of parental centrioles was not statistically affected compared with control cells (Fig 1A and Supplementary Fig S1I). These results indicate that the ectopic centrin foci in RBM14-depleted cells likely represent amplification of procentrioles or procentriole-like premature structures, or both, rather than multiple rounds of centriole duplication. We also noted that most of these structures contained other centriolar proteins such as CPAP (Hung et al, 2000) (65 ± 3%, n = 150 in triplicate; Supplementary Fig S1J) and CP110 (Chen et al, 2002) (55 ± 3%, n = 150 in triplicate; Supplementary Fig S1J) that constitute the middle-distal parts of centrioles. Consistently, we found significant increase in the number of CPAP and CP110 foci in the cells depleted of RBM14 (23 ± 2% compared with 0% in control cells, 30 ± 2% compared with 6 ± 2% in control cells, respectively; for both cases, P < 0.01, n = 90 in triplicate; Fig 1C and Supplementary Fig S1K). Furthermore, we observed ectopic foci of acetylated tubulin in the cells depleted of RBM14 (11 ± 1% compared with 1 ± 1% in control cells, P < 0.01, n = 90 in triplicate; Fig 1D) and also found that some of ectopic centrin-containing structures appeared to incorporate acetylated tubulin (26 ± 2%, n = 150 in triplicate; Supplementary Fig S1J), suggesting that they contain the centriolar microtubules. We next tested whether the ectopic centrin-containing structures are functional enough to assemble PCM. Upon depletion of endogenous RBM14, the ectopic centrin-containing structures efficiently accumulated PCM proteins such as γ-tubulin and Cep192 (Stearns et al, 1991; Zheng et al, 1991; Gomez-Ferreria et al, 2007; Zhu et al, 2008) (39 ± 2% and 41 ± 2%, respectively, n = 150 in triplicate; Supplementary Fig S1J). In line with this, we found significant increase in the number of ectopic PCM formation (22 ± 3% compared with 4 ± 1% in control cells for γ-tubulin, P < 0.01, n = 90 in triplicate; 22 ± 2% compared with 3 ± 2% in control cells for Cep192, P < 0.05, n = 90 in triplicate; Fig 1E and Supplementary Fig S1L). Based on these observations, we refer to the ectopic structures containing centriolar proteins and induced in RBM14-depleted cells as "centriolar protein complexes" in this article. Cytoplasmic RBM14 can inhibit centriole amplification in cells arrested in S phase To confirm that the appearance of ectopic centriolar protein complexes specifically results from loss of RBM14 and also to identify the region of RBM14 required for its function, we tested whether full-length (FL) and/or fragments of RBM14 can rescue the phenotype provoked by treatment with siRNA against the 3′ UTR targeting solely endogenous RBM14. We found that expression of the C-terminal half of RBM14 (RBM14[C]) lacking the RNA-recognition motif (RRM) as well as RBM14 FL markedly suppressed the amplification of centriolar protein complexes in the cells depleted of endogenous RBM14 (Fig 2A and B, and Supplementary Fig S2A). Figure 2. RBM14 can suppress centriole amplification in cells arrested in S phase A. U2OS cells or U2OS cells expressing FLAG–RBM14 full length (FL, aa1–669), N-terminal fragment (RBM14[N], aa1–150) or C-terminal fragment (RBM14[C], aa151–669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test). B. Schematic of full-length, truncated mutants and NES-fused and PACT-fused RBM14 proteins used in this figure. C, D. Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold). Download figure Download PowerPoint Next, to further investigate the function of RBM14 in normal centriole formation or centriole re-duplication in cells arrested in S phase, we ectopically expressed RBM14 FL and RBM14[C] in U2OS cells. Although overexpression of RBM14 had only a minor effect on normal centriole duplication and PCM assembly (Supplementary Fig S2B and C) or formation of multiple procentrioles induced by overexpression of Plk4, STIL or HsSAS-6 (Supplementary Fig S2D), we found that it significantly inhibited centriole amplification in cells treated with hydroxyurea (HU) to prolong S phase, judged by the frequency of cells with more than 4 centrin foci (26 ± 3% in cells expressing RBM14 FL, 27 ± 2% in cells expressing RBM14[C] compared with 53 ± 4% in control cells, P < 0.05, n = 90 in triplicate; Fig 2C). We noted in addition that ectopically expressed RBM14 FL proteins mainly existed in the nucleus, but also localized in the cytoplasm as is the case for the endogenous proteins (Fig 2C and Supplementary Fig S2A, E and F). By contrast, we found that RBM14[C] could occasionally localize in the vicinity of centrioles during interphase (14.4 ± 2.9% compared with 2.2 ± 1.1% of RBM14 FL and 1.1 ± 1.1% of endogenous RBM14, n = 90; Fig 2C and Supplementary Fig S2E), implying that this region may bind to a centriolar protein. These observations prompted us to investigate where RBM14 could act on centriole biogenesis. To address this, we manipulated the localization of RBM14 by fusing NES (nuclear export signal) or PACT domain, a centrosomal targeting motif, to the C-terminal end of RBM14 (Fig 2B). Intriguingly, we found that when overexpressing RBM14-NES which localized to the cytoplasm, but seemingly not to centrioles (only 2.2 ± 1.1% of RBM14-NES in the vicinity of centrioles during interphase, n = 90; Fig 2D), HU-induced centriole amplification was efficiently suppressed as is the case for overexpression of the native full-length protein (21 ± 1% in cells expressing RBM14-NES compared with 28 ± 3% in cells expressing RBM14, P < 0.05, n = 90 in triplicate; Fig 2D). On the other hand, overexpression of RBM14-PACT localizing to centrioles (33.3 ± 3.8%, n = 90; Fig 2D) and in the cytoplasm also suppressed the centriole amplification, but not more effectively (34 ± 4%, n = 90 in triplicate; Fig 2D), suggesting that the centriolar loading of RBM14 itself seems not to be essential for its ability to suppress centriole amplification. Furthermore, we established that expression of RBM14-NES suppressed the phenotype provoked by depletion of endogenous RBM14 (Supplementary Fig S2G). Overall, these data lead us to propose that cytoplasmic localization of RBM14 is crucial for its function to suppress amplification of centrioles or ectopic centriolar protein complexes. RBM14 interacts with STIL and prevents a complex formation of STIL and CPAP We next confirmed by co-immunoprecipitation experiments with the endogenous proteins that RBM14 is a bona fide STIL-binding protein in vivo (Fig 3A and Supplementary Fig S3A). On the other hand, we could not detect interaction between endogenous STIL and CPAP proteins in these experiments. Moreover, yeast two-hybrid, GST pull-down and co-immunoprecipitation assays using full-length and fragments of STIL and RBM14 established that the N-terminal region of STIL (STIL[N]) directly bound to the C-terminal region of RBM14, which is crucial for the ability of RBM14 to suppress the formation of ectopic centriolar protein complexes (Fig 3B and C, and Supplementary Fig S3B–D). Furthermore, using GST pull-down assays with several deletion mutants of RBM14[C], we determined that the TRBP (thyroid hormone receptor-binding protein)/Ncoa6-interacting domain (307–584 aa) (Iwasaki et al, 2001) is responsible for STIL–RBM14 binding (Fig 3C and Supplementary Fig S3E and F). This domain is composed of a serine/alanine/glycine/tyrosine-rich region and thought to be a protein–protein binding domain of RBM14. Consistently, functional analysis revealed that expression of the RBM14 mutant proteins that lack the TRBP-interacting domain did not efficiently rescue the phenotype provoked by depletion of endogenous RBM14 (Supplementary Fig S3G), suggesting that direct binding of RBM14 to STIL through the TRBP-interacting domain is required for the function of RBM14 suppressing ectopic formation of centriolar protein complexes. These data prompted us to examine whether this interaction influences the expression levels or centriolar localization of STIL, or both. However, both immunofluorescence and Western blot analyses in RBM14-depleted U2OS cells showed that fluctuating expression at centrioles and total expression levels of STIL across the cell cycle (Tang et al, 2011; Arquint et al, 2012; Vulprecht et al, 2012) seemed comparable to the control (Fig 3D and Supplementary Fig S4A). There was also no alteration in the localization of STIL at the proximal end of procentrioles (Tang et al, 2011; Arquint et al, 2012; Vulprecht et al, 2012) (G. Shiratsuchi, D. Kitagawa, unpublished observation). However, we noted that relatively small ectopic centrin foci, but not seemingly mature centrin foci, tended to contain STIL signals during mitosis (9 ± 3% for RBM14-depleted cells, 1 ± 1% for control cells, P < 0.05, n = 90 in triplicate; Fig 3D), implying that STIL could localize to premature centriolar protein complexes only transiently. Based on these data, we postulated that the binding of RBM14 to STIL might affect the function of STIL in centriole formation. Given that STIL[N] contained the region responsible for CPAP-binding (231–781 aa) (Tang et al, 2011), we assumed that RBM14 may disrupt the interaction between STIL and CPAP. To address this, we conducted in vitro pull-down assay to test whether RBM14[C] and the STIL-binding region of CPAP, CPAP[SBD], compete with each other for binding to STIL[N]. We found this to be the case, supporting the model in which RBM14 prevents the formation of STIL/CPAP complex (Fig 3E). Furthermore, we found that addition of RBM14 FL or RBM14[C], but not RBM14[N], efficiently dampened the complex formation of STIL and GFP-CPAP in U2OS cells (Fig 3F). These findings are in line with the fact that the C-terminal region of RBM14 is responsible for STIL binding (Fig 3B and C, and Supplementary Fig S3). Importantly, we revealed, using siRNA-based double knockdown experiments, that the formation of ectopic centrin foci by RBM14 depletion depends on CPAP and STIL (Fig 3G). Moreover, to further confirm the biological relevance of the complex formation of STIL and CPAP in this process, we tested whether expression of STIL mutants, STIL[N] and STIL[CBD], that contain CPAP-binding domain (CBD), but lack the conserved STAN motif, could act in a dominant-negative manner to inhibit the formation of the ectopic centriolar protein complexes in RBM14-depleted cells. Accordingly, we found that this was indeed the case (Supplementary Fig S4B). Overall, these findings lead us to propose that the interaction of RBM14 with STIL suppresses the inherent ability of the STIL/CPAP complex for the ectopic formation of centriolar protein complexes. Figure 3. RBM14 interacts with STIL and prevents a complex formation of STIL and CPAP HeLa cells immunoprecipitated with control IgG or STIL antibodies. Soluble cytosolic fractions (input) and immunoprecipitates (IPs) were analyzed by Western blotting using RBM14, STIL or CPAP antibodies. GST pull-down assay testing interactions between purified STIL[N] (˜5 μg, aa1–1018) and GST-RBM14 [N] or [C]. The asterisks indicate non-specific bands. Schematic of our analyses of Y2H, GST pull-down and co-immunoprecipitation of the interaction between RBM14 and STIL (see also Supplementary Fig S3). Brackets indicate the fragments tested in this study, and the interaction detected is shown with arrows. A previous study reported that the C-terminus of CPAP interacts with the fragment of STIL aa231–781, as indicated (Tang et al, 2011). Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin-2 (green) and STIL (magenta). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess STIL foci co-localized with centrin foci. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01 (one-tailed t-test). In vitro competitive binding assay. GST pull-down experiment was performed as in (B), with purified STIL[N] and GST-RBM14[C] in the presence of the indicated amount of purified His-CPAP[SBD], His-tagged STIL-Binding Domain of CPAP. The fraction of STIL[N] bound to GST-RBM14[C] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N-terminal region of STIL. Input materials were analyzed by Western blotting using the STIL or CPAP antibodies. The precipitated GST-RBM14[C] was analyzed by SDS–PAGE, stained with SimplyBlue™ Safe (Invitrogen). Interaction between STIL and GFP-CPAP in the presence of FLAG-RBM14 FL or fragments. U2OS cells expressing GFP-CPAP were transfected with empty vector as a control, FLAG-RBM14[N], FLAG-RBM14[C] or FLAG-RBM14 FL constructs, immunoprecipitated with STIL antibodies, and the resulting IPs were analyzed by Western blotting using STIL, CPAP or FLAG antibodies. Quantification of relative protein amounts of co-immunoprecipitated GFP-CPAP in each STIL-IP fraction, normalized with the amount of STIL precipitated, is shown below the panels. Means ± SEM were calculated from three independent experiments. **P < 0.01 (one-tailed t-test). Note that we did not find any significant effect of exogenous expression of RBM14 full-length protein or deletion mutants on the expression levels of GFP-CPAP or endogenous STIL proteins in this experiment. U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta). Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Representative mitotic cells treated with siRNAs against RBM14 alone, RBM14 + STIL or RBM14 + CPAP are shown below. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Download figure Download PowerPoint The ectopic formation of centriolar protein complexes occurs in the cytoplasm in a HsSAS-6-independent manner Next, to test the dependency of a cartwheel structure on the formation of ectopic centriolar protein complexes, we conducted RNAi-mediated reduction of HsSAS-6 in the RBM14-depleted cells. Strikingly, this analysis revealed that the formation of ectopic centrin foci was mostly independent of the existence of HsSAS-6 (Fig 4A and Supplementary Fig S5A and B). Similarly, depletion of Plk4 did not affect the amplification of centriolar protein complexes (Supplementary Fig S5C). These results suggest that the amplification of centriolar protein complexes induced by RBM14 dep
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