Regular Article an X-Chromosome RNA-Binding Motif Protein (RBMX

Chromosome Science 14: 17-21, 2011 Lee et al. 17

Regular Article

An X-chromosome RNA-binding motif protein (RBMX) is required for proper kinetochore formation

Mei Hann Lee, Linyen Lin, Hideaki Takata, Akihiro Morimoto, Susumu Uchiyama, Sachihiro Matsunaga and Kiichi Fukui

Abstract 2007; Tanaka 2008). The mammalian kinetochore is a small and yet an elaborate structure, providing physical RBMX is an RNA-binding motif protein identified as a attachment to the microtubules, force generation and component of purified human metaphase chromosomes. spindle assembly checkpoint (SAC) signaling that delays We have analyzed the functional significance of RBMX anaphase onset until all chromosomes are attached to the by electron microscopy (EM) in combination with RNA spindle (Cheeseman and Desai 2008). This intricate cellular interference (RNAi). We revealed that depletion of RBMX machinery comprises of more than 120 components (Ohta results in the accumulation of mitotic cells. EM investi- et al. 2010), and an ever-increasing number of proteins are gation further showed that RBMX depletion leads to an being implicated in, and changing our understanding of increase in the fuzzy ball structure that is reminiscent of the kinetochore function and structure. the prekinetochore. It is most probably due to the result Early electron microscopic (EM) studies with of disruption at early stages of kinetochore maturation, conventional chemical fixative methods established so that kinetochore differentiation into the authentic tri- the classical trilaminar model of kinetochore structure laminar morphology is disturbed. The present EM data (Brinkley and Stubblefield 1966; Comings and Okada illuminated the importance of RBMX in kinetochore for- 1971). The inner kinetochore forms the interface with mation for subsequent stable microtubules interaction. chromatin; the middle kinetochore is a less electron-dense interzone; and the outer kinetochore is a flexible network Keywords: that interacts with multiple kinetochore microtubules RBMX (RNA-binding motif protein), hnRNP G (hetero- (Dong et al. 2007). In the absence of microtubule geneous nuclear ribonucleoprotein G), kinetochore, EM, attachment, a moderately dense fibrillar corona extended RNA interference from the outer kinetochore is visible (Cheeseman and Desai 2008; McEwen and Dong 2010). Numerous studies have shown that kinetochores are formed transiently on Introduction the centromere during mitosis (Roos 1973; Rieder 1982), suggesting that kinetochores underwent an assembly/ During mitosis, two complete sets of chromosomes disassembly cycle (Brenner et al. 1981; He and Brinkley, carrying identical genetic information are apportioned to a 1996). Correlative light and electron microscopy (CLEM) pair of daughter cells. Error-free sister chromatid separation reveals that kinetochores are visible on the surface of the is orchestrated by the attachment of the sister kinetochores primary constrictions as roughly circular patches of finely of each chromosome to microtubules emanating from fibrillar materials as cells enter the prophase and gradually opposite spindle poles (O’Connell and Khodjakov differentiate into the trilaminar morphology that is visible until anaphase (Roos 1973). hnRNP G (heterogeneous nuclear ribonucleoprotein G) is implicated in the splicing control of several pre- Mei Hann Lee, Hideaki Takata, Akihiro Morimoto, mRNAs (Glisovic et al. 2008), and hnRNP G promotes Susumu Uchiyama, Sachihiro Matsunaga*, Kiichi Fukui (*) the expression of tumor-suppressor Txnip and protects Department of Biotechnology, Graduate School of Engineering, the fidelity of DNA end-joining activity (Shin et al. Osaka University, GSE Common East 7 F, 2-1 Yamadaoka, Suita, 2008). The relatively low abundance hnRNP G protein is Osaka 565-0871, Japan. unique among hnRNPs (Kanhoush et al. 2010) for being Email address: [email protected] glycosylated (Soulard et al. 1993). In human cells, the gene Linyen Lin coding for hnRNP G is located in the X chromosome, and Research Center for Ultra-High Voltage Electron Microscopy, therefore it is also known as RBMX (RNA-binding motif Osaka University. 7-1, Mihogaoka, Ibaraki, Osaka 567-0047, Japan protein, X chromosome). RBMX is subject to X inactivation *Present address. (Soulard et al. 1993), and is critical for proper neural Department of Applied Biological Science, development of zebrafish and frog embryos (Tsend-Ayush Faculty of Science and Technology, Tokyo University of Science et al. 2005; Dichmann et al. 2008). Multiple processed 2641 Yamazaki, Noda, Chiba 278-8510, Japan copies of RBMX are present in the human genome, 18 RBMX is required for kinetochore formation suggesting that RBMX has multiple roles (Lingenfelter et transfection for analyses. al. 2001). Our previous efforts in elucidating chromosome higher order structure based on their constituent proteins Immunoblotting and gel electrophoresis enabled us to identify over 200 proteins by proteome analysis of human metaphase chromosomes (Uchiyama et Cells (siRNA or mock-transfected) grown in 24-well al. 2005). RBMX was identified as an abundant protein in plates were collected and lysed in 2х Laemmli Sample isolated chromosomes, suggesting its function in mitosis Buffer (Santa Cruz Biotechnology) with equal amounts (Fukui and Uchiyama 2007; Takata et al. 2007). However, of PBS buffer. Protein extracts were fractionated on 10% the function of RBMX in mitotic chromosomes has not polyacrilamide gels and then transferred onto PVDF been well investigated. membranes. The immunoblots were blocked with 1% We have now shown that RBMX is essential for mitotic BSA-TBST (0.1% Tween 20, 25 mM Tris-HCl･pH 7.4, 137 progression in human cells, suggested that RBMX is mM NaCl, 25 mM KCl) and labeled with the primary and involved in kinetochore formation. This conclusion is secondary antibodies. The immunoreactive protein bands reinforced by ultrastructural analysis of kinetochores in were detected by NBT/BCIP solution (Roche) diluted in RBMX-depleted cells. RBMX is required for kinetochore AP buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 1 mM plate development and stable microtubule attachment; MgCl2). prerequisites for accurate chromosome congression and segregation. Fluorescence microscopy HeLa cells grown on coverslips were transfected with the target siRNA as above, fixed with 4% PFA (para- Materials and Methods formaldehyde) diluted with PBS (phosphate buffered saline, Cell culture pH 7.4) for 15 min at 37 °С, and stained with Hoechst 33342 (Sigma) for mitotic index calculation. HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL) supplemented with Electron microscopy 10% fetal-bovine serum (FBS; Equitech-Bio) at 37°С and 5% CO2. Sample preparation and EM analyses were based on those of Lee et al. (2011). In brief, HeLa cells grown on Antibodies plastic coverslips (mono-layer) were fixed for 1 h in 3% glutaraldehyde and 0.2% tannic acid in PBS buffer at room For immunoblotting, primary antibodies were goat temperature. Post-fixation was in 2% OsO4 for 20 min. polyclonal anti-RBMX antibody (1:1000; Santa Cruz The cells were dehydrated through an increasing ethanol Biotechnology) and mouse monoclonal anti-b-actin series and infiltrated with epoxy resin (Quetol 812). The antibody (1:10000; Sigma). Secondary antibodies were resins were polymerized at 37 °С for 12 h, 45 °С for 12 h alkaline phosphatase anti-mouse IgG (1:2000; Vector and 60 °С for 48 h. Plastic coverslips were removed, and Laboratories) and alkaline phosphatase anti-goat IgG cells of interest embedded in the resin were chosen under (1:2000; Vector Laboratories). an optical microscope and trimmed to ~ 1.0 mm2. Samples were cut into 70-80 nm thick serial sections, parallel with siRNA methods the coverslip direction, with an ultramicrotome equipped with a diamond knife (ULTRACUT E, Reichart-Jung). The HeLa cells were transfected at a final concentration of 100 sections were stained with uranyl acetate and lead citrate nM with RBMX-siRNA (5’-uca aga gga uau agc gau att-3’) and examined by a transmission electron microscope using Lipofectamine 2000 according to the manufacturer’s (JOEL, JEM-1200EX). instructions. Cells transfected with Lipofectamine alone For EM analyses, cells that apparently aligned at were used as a control. Cells were collected 48h post- metaphase plate were chosen for control, and RBMX RNAi cells were chosen based on the phenotypes of poor chromosome alignment. Alternatively, colcemid treated cells were chosen randomly both in the control and RBMX RNAi. All kinetochores observed were included in the analyses regardless of their appearance. Only a few sections, containing chromosome-rich regions, were examined for individual cells. As the boundary between individual chromosomes is not obvious, and sister kinetochore appearances can sometimes

Figure 1. Mitotic defects in RBMX disruption. A De- pletion of RBMX by RNAi treatment. β-actin was used as loading control. RBMX has a molecular weight of 43 kDa. B Low magnification electron micrograph of a control cell. C Typical phenotype of RBMX disrupted cells. Scale bar: 10 µm. Lee et al. 19

Figure 2. Kinetochores in the absence of microtubules with colcemid treatment. A Well-formed sister kinetochores with fibrous coronas extending from the outer plates, the major features in the control. B Poorly-formed kinetochore in RBMX RNAi cell. C Fuzzy ball in RBMX depleted cell. A-C Red and yellow arrows represent outer and inner kinetochores, respectively. Blue arrows denoted fuzzy balls. All micrographs are in the same magnification. Adjacent serial sections were indicated as i-ii. Scale bar: 500 nm. D Quantitative measurement of the kinetochores. 34 kinetochores from 4 control cells and 31 kinetochores from 8 RBMX RNAi cells were examined. be seen differently depending on kinetochore fiber plates are not always visible in colcemid treatment (Roos attachment, kinetochores were analyzed individually rather 1973), kinetochores with an electron-dense outer layer than as a pair of kinetochores of a chromosome. Several are considered well-formed. In the control, nearly 90% of adjacent serial sections were analyzed to classify individual the kinetochores were well-formed (Fig. 2A). In contrast, kinetochores, because kinetochore morphology varies even nearly 70% of the kinetochores in RBMX RNAi cultures between adjacent serial sections. Therefore kinetochore is failed to construct the layered structure and appeared classified as trilaminar once the canonical layered structure as fuzzy balls (Fig. 2B, 2C), although outer plates were is visible in any of the adjacent serial sections for an constructed to some degree in some kinetochores (Fig. 2B). individual kinetochore, even if the structure is rather fuzzy Next, we examined whether the kinetochore aberrations in other sections. affected microtubules interaction. We imaged adjacent serial sections of 5 control cultured cells (Fig. 1B) and 11 Results RBMX-depleted cultured cells (Fig. 1C). The kinetochores were again classified based on our classification scheme, Mitotic progression was impaired by RBMX depletion. and each group was further divided into two categories, To assess the functional roles of RBMX in mitosis, we i.e. with or without microtubule attachment. Inner plates employed an RNAi-mediated gene-silencing approach to are visible in most of the well-formed kinetochores. Fig. knockdown RBMX from the cells. Immunoblot analysis of 3A shows the frequency of kinetochore disorganization HeLa cultures subjected to RBMX siRNA treatment for 48 in RBMX RNAi cultures. 58 and 56 kinetochores were hours revealed a decline in the expression level of RBMX examined in the control and RBMX RNAi cultures, of less than 20% when compared with mock transfected respectively. In the control, robust microtubules cells (Figure 1A). Mitotic index was increased to more attachment (white arrowheads) was observed, regardless than 3-fold (13.1±3.3%) of the control (4.4±0.2%). A of the kinetochore structures (Fig. 3A-F). When RBMX high degree of alignment defects (74.3±6.8%, compare to is knockdowned, kinetochore-microtubule interactions 4.7±4.6% in the control) was observed as we assayed for were significantly reduced, especially for poorly-formed or the mitotic profiles in cells lacking RBMX, as shown in the fuzzy ball kinetochores (Fig. 3A, 3G-L). These data indicate lower magnification electron micrographs (Figure 1B, C). that RBMX is essential for proper kinetochore formation for subsequent stable microtubule interactions to achieve RBMX depletion compromises kinetochore formation chromosome congression. Chromosome congression defects are a common feature of aberrant microtubule association. Often, irregular Discussion kinetochore formation couples with chromosome-to- spindle attachment defects. Thus, we evaluated whether RBMX has been identified in screenings of mitotic kinetochores assemble properly in RBMX RNAi cells with chromosome components in both human (Uchiyama et electron microscopy using conventional chemical fixation. al. 2005; Takata et al. 2007) and DT40 cell lines (Ohta et First, we treated the cells with colcemid to examine the al. 2010), suggesting its involvement in the mitotic events. kinetochore structure. 34 kinetochores from 4 control In this study, we found that cells lacking RBMX show cells and 31 kinetochores from 8 RBMX RNAi cells were particularly aberrant chromosome congression, suggesting examined (Fig. 2). Kinetochores were classified into three that they are defective with respect to microtubule capture. groups, i.e. well-formed (Class 3), poorly-formed (Class EM studies support this conclusion. Authentic trilaminar 2), and fuzzy ball (Class 1), based on the classification kinetochores are notably rarer in RBMX RNAi cultures. scheme we have proposed (Lee et al. 2011). Because inner Fuzzy ball structures, resembling the prekinetochores 20 RBMX is required for kinetochore formation

Figure 3. Frequency of kinetochore disorganization in RBMX RNAi cells revealed by high magnification electron micrographs. A Kinetochores in control and RBMX RNAi treatments were classified as well-formed, poorly-formed, or fuzzy ball, based on their structure. Microtubule interactions were determined for each group. 58 and 56 kinetochores were examined in the control and RBMX RNAi cultures, respectively. B-F Kinetochores in control cells. B Control trilaminar kinetochores with microtubule attachment, classified as well-formed. C Poorly-formed kinetochore with microtubule attachment. D Poorly-formed kinetochore without microtubule attachment. E Fuzzy ball with microtubule association. F Fuzzy ball. No microtubules were detected. G-L Kinetochores in RBMX RNAi cells. G Well-formed kinetochore with microtubule attachment. H Well-formed kinetochore without microtubule attachment. I Kinetochore with less distinct outer plate (poorly-formed). End-on microtubules attachment was observed. J Poorly-formed kinetochore without microtubules interaction. K Fuzzy ball with microtubule interaction. L Fuzzy balls without microtubule association. B-L Red and yellow arrows represent outer and inner kinetochores, respectively. Blue arrows denoted fuzzy balls. White arrowheads indicated microtubules. All micrographs are in the same magnification. Adjacent serial sections were indicated as i-v. Scale bar: 500 nm.

(Roos 1973; Lee et al. 2011) often observed in early mitotic 3A). stages, suggest that kinetochore maturation is highly The observation of well-formed kinetochore in RBMX disrupted. Normally, most of the kinetochores interact with RNAi cells was not surprising, as DeLuca et al. (2005) microtubules after nuclear envelope breakdown, even when showed that trilaminar kinetochores were observed even the trilaminar structure has not been fully constructed when Hec1-Nuf2, the major components constructing (data not shown; Roos 1973). Only a few kinetochores in the outer kinetochore plates were depleted. We think that RBMX RNAi cells associate with microtubules; those with it was because RBMX was knockdown but not knockout a particularly low population appeared as fuzzy balls (Fig. from the cells, therefore some kinetochores may develop Lee et al. 21 even the proteins level is very low in that cell. Fuzzy ball The outer plate in vertebrate kinetochores is a flexible network structures observed in the control may be derived from with multiple microtubule interactions. Nat Cell Biol 9:516-522 several reasons: differences in kinetochore maturation, Fukui K, Uchiyama S (2007) Chromosome protein framework from microtubule interactions and angle of the kinetochores. proteome analysis of isolated human metaphase chromosomes. Chem Rec 7:230-237 Well-formed kinetochores increased in colcemid treatment, Glisovic T, Bachorik JL, Yong J, Dreyfuss G (2008) RNA-binding suggested that differences in kinetochore maturation and proteins and post-transcriptional gene regulation. FEBS Lett microtubule association contributed to some degree in the 582:1977-1986 fuzzy ball appearance in the control. These may also be the He D, Brinkley BR (1996) Structure and dynamic organization of cen- case in RBMX RNAi cells, because fuzzy ball structures tromeres/prekinetochores in the nucleus of mammalian cells. J were slightly decreased, although well-formed kinetochores Cell Sci 109:2693-2704 remained low, and microtubule attachments were fewer Kanhoush R, Beenders B, Perrin C, Moreau J, Bellini M, Penrad-Mo- without colcemid treatment. It was shown by Roos bayed M (2010) Novel domains in the hnRNP G/RBMX protein (1973) that kinetochores appeared as fuzzy balls in para- with distinct roles in RNA binding and targeting nascent tran- equatorial sections. Therefore, fuzzy ball in the control scripts. Nucleus 1:109-122 Lee MH, Lin L, Ilma Equilibrina, Uchiyama S, Matsunaga S, Fukui K may also caused by kinetochore angle during sectioning. (2011) PHB2 (ASURA) is required for kinetochore assembly and Nevertheless, the fuzzy balls in the control are relatively subsequent chromosome congression. Acta Histochem Cytochem low, and the percentages of well-formed kinetochores in 44:247-258 colcemid treated cells were consistent with the previous Lingenfelter PA, Delbridge ML, Thomas S, Hoekstra HE, Mitchell MJ, report (DeLuca et al. 2005). Graves JA, Disteche CM (2001) Expression and conservation of In conclusion, we have shown that RBMX plays critical processed copies of the RBMX gene. Mamm Genome 12:538-545 role in proper kinetochore formation where subsequent McEwen BF, Dong Y (2010) Contrasting models for kinetochore stable microtubules attachment is attained. Failure to do so microtubule attachment in mammalian cells. Cell Mol Life Sci induced mitotic arrest (data not shown) with high degree 67:2163-2172 of alignment defects, resulted in the increasing of mitotic O’Connell CB and Khodjakov AL (2007) Cooperative mechanisms of mitotic spindle formation. J Cell Sci 120:1717-1722 index. Detailed characterization of the mechanism is under Ohta S, Bukowski-Wills JC, Sanchez-Pulido L, Alves Fde L, Wood L, investigation and will be reported elsewhere. Chen ZA, Platani M, Fischer L, Hudson DF, Ponting CP, Fukagawa T, Earnshaw WC, Rappsilber J (2010) The protein composition of mitotic chromosomes determined using multiclassifier combinato- Acknowledgement rial proteomics. Cell 142:810-821 Rieder CL (1982) The formation, structure, and composition of the Some of the present experiments were undertaken mammalian kinetochore and kinetochore fiber. Int Rev Cytol 79:1- at the Research Center for Ultrahigh Voltage Electron 58 Microscopy, Osaka University. We are especially grateful for Roos UP (1973) Light and electron microscopy of rat kangaroo cells the excellent technical support of Mr. Toshiaki Hasegawa in mitosis. II. Kinetochore structure and function. Chromosoma 41:195-220 and Dr. Tomoki Nishida. We are also indebted to Associate Shin KH, Kim RH, Kim RH, Kang MK, Park NH (2008) hnRNP G Prof. Sumire Inaga and Dr. Tetsuo Katsumoto from the elicits tumor-suppressive activity in part by upregulating the ex- Faculty of Medicine, Tottori University, for their helpful pression of Txnip. Biochem Biophys Res Commun 372:880-885 advice and for their electron microscopy protocols. This Soulard M, Della Valle V, Siomi MC, Piñol-Roma S, Codogno P, Bauvy work was supported by the Grants-in-Aid for Scientific C, Bellini M, Lacroix JC, Monod G, Dreyfuss G, Larsen C-J (1993) Research from the Ministry of Education, Culture, Sports, hnRNP G sequence and characterization of a glycosylated RNA- Science and Technology of Japan (MEXT, No. 21248040) binding protein. Nucleic Acids Res 21:4210-4217 to K.F. M.H.L. would like to thank MEXT for fellowship Takata H, Uchiyama S, Nakamura N, Nakashima S, Kobayashi S, Sone support. T, Kimura S, Lahmers S, Granzier H, Labeit S, Matsunaga S, Fukui K (2007) A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosome-associated proteins. Genes Cells 12:269- References 284 Tanaka TU (2008) Bi-orienting chromosomes: acrobatics on the mi- Brenner S, Pepper D, Berns MW, Tan E, Brinkley BR (1981) Kineto- totic spindle. Chromosoma 117:521-533 chore structure, duplication, and distribution in mammalian cells: Tsend-Ayush E, O’Sullivan LA, Grützner FS, Onnebo SM, Lewis RS, analysis by human autoantibodies from scleroderma patients. J Delbridge ML, Marshall Graves JA, Ward AC (2005) RBMX gene Cell Biol 91:95-102 is essential for brain development in zebrafish. Dev Dyn 234:682- Brinkley BR, Stubblefield E (1966) The fine structure of the kineto- 688 chore of a mammalian cell in vitro. Chromosoma 19:28-43 Uchiyama S, Kobayashi S, Takata H, Ishihara T, Hori N, Higashi T, Cheeseman IM, Desai A (2008) Molecular architecture of the kineto- Hayashihara K, Sone T, Higo D, Nirasawa T, Takao T, Matsunaga S, chore-microtubule interface. Nat Rev Mol Cell Biol 9:33-46 Fukui K (2005) Proteome analysis of human metaphase chromo- Comings DE, Okada TA (1971) Fine structure of kinetochore in In- somes. J Biol Chem 280:16994-17004 dian muntjac. Exp Cell Res 67:97-110 DeLuca JG, Dong Y, Hergert P, Strauss J, Hickey JM, Salmon ED, McE- wen BF (2005) Hec1 and Nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites. Mol Biol Cell 16:519-531 Dichmann DS, Fletcher RB, Harland RM (2008) Expression cloning in Xenopus identifies RNA-binding proteins as regulators of embryo- genesis and Rbmx as necessary for neural and muscle development. Dev Dyn 237:1755-1766 Dong Y, Vanden Beldt KJ, Meng X, Khodjakov A, McEwen BF (2007)