Methylation of Ribosomal Protein S10 by Protein-arginineMethyltransferase 5 Regulates Ribosome Biogenesis*□S

Received for publication, January 14, 2010, and in revised form, February 11, 2010 Published, JBC Papers in Press, February 16, 2010, DOI 10.1074/jbc.M110.103911

Jinqi Ren, Yaqing Wang, Yuheng Liang, Yongqing Zhang, Shilai Bao, and Zhiheng Xu1

From the Institute of Genetics and Developmental Biology, The Key Laboratory of Molecular and Developmental Biology, ChineseAcademy of Sciences, Beijing 100101, China

Modulationof ribosomal assembly is a fine tuningmechanismfor cell number andorgan size control.Many ribosomal proteinsundergo post-translational modification, but their exact rolesremain elusive. Here, we report that ribosomal protein s10(RPS10) is a novel substrate of an oncoprotein, protein-argininemethyltransferase 5 (PRMT5). We show that PRMT5 interactswith RPS10 and catalyzes its methylation at the Arg158 andArg160 residues. Themethylation of RPS10 at Arg158 and Arg160

plays a role in the proper assembly of ribosomes, protein synthe-sis, and optimal cell proliferation. The RPS10-R158K/R160Kmutant is not efficiently assembled into ribosomes and is unsta-ble and prone to degradation by the proteasomal pathway. Innucleoli, RPS10 interacts with nucleophosmin/B23 and is pre-dominantly concentrated in the granular component region,which is required for ribosome assembly. The RPS10 methyla-tionmutant interacts weakly with nucleophosmin/B23 and failsto concentrate in the granular component region. Our resultssuggest that PRMT5 is likely to regulate cell proliferationthrough themethylation of ribosome proteins, and thus reveal anovel mechanism for PRMT5 in tumorigenesis.

Ribosomes are complex macromolecular machines com-posed of rRNAs and ribosomal proteins and are responsible forthe synthesis of polypeptide chains. Ribosome biogenesis isthus a critical process inextricably linked to cell growth andproliferation. The production of mature ribosomes competentto translate mRNAs is a highly coordinated multistep process(1). Post-translational modifications of ribosomal proteins,including phosphorylation, methylation, and acetylation, affecttheir characteristics and ribosome biogenesis. Increased phos-phorylation of ribosomal protein S6, for example, has beenshown to result in enhanced translation efficiency of specificmRNAs (2).Methylation is considered to be one of the most common

forms of post-translational modification of ribosomal proteins(3). It was shown in 1979 that 11 ribosomal proteins are likely tobemethylated in HeLa cells (4). Lhoest et al. (5) estimated fromin vivo labeling experiments that the yeast 60 S subunit contains

about 10methylation groups, whereas the 40 S subunit has 2–4methylation groups. The pattern of ribosomal protein methyl-ation appears to be similar in various eubacteria, but there is ahigher occurrence of argininemethylation in higher eukaryotes(3). Ribosomal protein S2 was the first arginine methylationsubstrate found for protein-arginine methyltransferase 3(PRMT3)2 (6, 7). Yeast cells lacking PRMT3 showed accumu-lation of free 60 S ribosomal subunits, resulting in an imbalancein the 40 S:60 S free subunits ratio (8). However, argininemeth-ylation of other ribosomal proteins, especially in highereukaryotes, and its biofunctions remain to be elucidated.Protein-arginine methyltransferases are enzymes that catalyze

the transfer of amethyl group from S-adenosylmethionine to argi-nine (9, 10). PRMTsare classified into twogroups.Although type IPRMTs catalyze the formation ofmonomethylarginine and asym-metric dimethylarginine (aDMA), type II enzymes catalyzemonomethylarginine and symmetric dimethylarginine (sDMA).Recently, accumulating evidence has revealed that argininemeth-ylation has important roles in many cellular processes, includingRNA processing, transcriptional regulation, signal transduction,DNA repair, and protein-protein interactions (9, 10). In humans,the PRMT family has been shown to contain around 11 methyl-transferases, designated as PRMT1–11 based on their primarysequence and substrate specificity (11).PRMT5 or Skb1Hs, a type II arginine methyltransferase was

originally cloned as a human homolog of Shk1 kinase binding 1(Skb1) in fission yeast (12). It was later reported to interact witha number of complexes and to be involved in a variety of cellularprocesses. PRMT5 methylates histones H2A, H3, and H4 toregulate gene expression (13–15), Smproteins B-B�, D1, andD3to promote their transfer to the SMN complex and enhancesmall nuclear ribonucleoprotein biogenesis (16–18), and SPT5to regulate its interactionwithRNApolymerase II thus control-ling transcriptional elongation (19).The biological functions of PRMT5 have been identified in

recent years. We have shown in fission yeast that Skb1 plays animportant role in regulating cell growth and polarity underhyperosmotic stress (20). We also found that the homolog ofPRMT5 in plants, SKB1, plays a role in methylation of histone

* This work was supported in part by the Ministry of Science and Technologyof China “973” program (2007CB947202/2006CB500701), “863” program(2006AA02Z173/Z140), and 2009DFA32450; the National Science Founda-tion of China (30970580/30725007/30670663/30870527/30670479); andthe Chinese Academy of Sciences (Bairen plan and KSCX1-YW-R-62/84).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4.

1 To whom correspondence should be addressed: 1 West Beichen Rd., Beijing,China 100101. Fax: 86-10-64840829; E-mail: zhxu@genetics.ac.cn.

2 The abbreviations used are: PRMT, protein-arginine methyltransferase;aDMA, asymmetric dimethylarginine; sDMA, symmetric dimethylarginine;GC, granular component; GFP, green fluorescent protein; GAPDH, glycer-aldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase;WT, wild-type; LRRK2, leucine-rich repeat kinase 2; siRNA, small interferingRNA; CHX, cycloheximide; SLE, systemic lupus erythematosus; shRNA,short hairpin RNA; EGFP, enhanced green fluorescent protein; DsRed, Dis-cosoma sp. red.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 17, pp. 12695–12705, April 23, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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H4R3 and is involved in the control of flowering time in Arabi-dopsis (21). In mammalian cells, overexpression of PRMT5induces hyperproliferation, whereas knockdown of PRMT5inhibits cell growth and proliferation (22–24). By forming com-plexes with hSWI/SNF chromatin-remodeling proteins BRGand BRM, PRMT5 can facilitate ATP-dependent chromatinremodeling and is required formuscle differentiation andmyo-genesis (25, 26). More importantly, PRMT5 protein levels areelevated in gastric cancer and various human lymphoid cancercells and mantle cell lymphoma clinical samples (23, 27).PRMT5 is also associated with tumorigenesis by suppressingthe transcription of the RB family and ST7 (suppressor oftumorigenicity 7) in leukemia and lymphoma cells (23, 28).Arginine methylation of p53 by PRMT5 also affects the targetgene specificity of p53, and PRMT5 depletion triggers p53-de-pendent apoptosis (29). Loss of E-cadherin is a hallmark of epi-thelial-mesenchymal transition. A recent report demonstratedthat PRMT5 knockdown results in elevated E-cadherin expres-sion, further indicating that PRMT5 is an oncoprotein (30).In this study, we identified RPS10 as a novel substrate for

PRMT5. PRMT5 interacts with RPS10 and methylates it onboth Arg158 and Arg160 residues. RPS10 is essential for cell pro-liferation and methylation. Furthermore, we provide evidencehere that PRMT5-mediated RPS10 methylation plays a role inthe interaction between RPS10 and B23, the localization ofRPS10 in the granular component (GC) region of the nucleolusand in the assembly of RPS10 into ribosomes.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

HEK293 and U2OS cells were cultured in Dulbecco’s modi-fied Eagle’s medium, whereas the hepatocellular carcinoma cellline Bel7402was grown in RPMI-1640, supplementedwith 10%fetal bovine serum.Cell transfectionwas performedusing Lipo-fectamine 2000 (Invitrogen).

Antibodies and Chemicals

�-Tubulin, FLAG antibody, and FLAG-agarose beads werepurchased from Sigma; Lamin B andMG132 fromCalbiochem;rabbit SYM11 polyclonal antibody fromUpstate BiotechnologyLtd.;Myc tagmonoclonal antibody, clone PL14 fromMBLLtd.;mouseGFP (clone B-2) andGAPDHantibody fromSantaCruz.RPS10 antiserumwas raised in mice using purified humanHis-tagged RPS10 as the immunizing antigen. To prepare the His-tagged RPS10 protein, pET-30a-RPS10 was transformed intoEscherichia coli, induced by 0.4mM isopropyl 1-thio-�-D-galac-topyranoside at 25 °C overnight, and purified by nickel-nitrilo-triacetic acid-agarose (Qiagen).

Plasmids

The RPS10 gene was amplified by PCR from human cDNAlibrary using primers 5�-CGGATATCGCCACCATGTTGAT-GCCTAAGAAG-3� and 5�-CGGGATCCCCCTGAGGTGG-CTGACCACG-3�, and inserted into EcoRV and BamHI sites ofpcDNA3.1/Myc-His(�) B (Invitrogen) andpET-30a(�) (Nova-gen). Glutathione S-transferase (GST) fusion RPS10 plasmidwas subcloned in pGEX-4T-2 (Amersham Biosciences) by PCR

and inserted in the BamHI/SalI sites (primers used: 5�-CGGG-ATCCACCATGTTGATGCCTAAGAAG-3� and 5�-CCGCT-CGAGTTACTGAGGTGGCTGACCACG-3�). GAR deletionof the GST fusion RPS10 (�GAR) plasmid was constructed byPCR using the following primers: 5�-CGGGATCCACCATGT-TGATGCCTAAGAAG-3�, and 5�-ACGCGTCGACTTAGA-ATTCGGTTGCTGAC-3�, and inserted into the BamHI/SalIsite in pGEX-4T-2. Methylation mutants of GST fusion RPS10plasmid were constructed by annealing different synthesizedoligos and inserting into the EcoRI/SalI site of pGEX-4T-2;R158K, 5�-AATTCCAGTTTAGAGGCGGATTTGGTAAG-GGACGTGGTCAGCCACCTCAGTAAC-3� and 5�-TCGAG-TTACTGAGGTGGCTGACCACGTCCCTTACCAAATCC-GCCTCTAAACTGG-3�; R160K, 5�-AATTCCAGTTTAGA-GGCGGATTTGGTCGTGGAAAGGGTCAGCCACCTCA-GTAAC-3� and 5�-TCGAGTTACTGAGGTGGCTGACCCT-TTCCACGACCAAATCCGCCTCTAAACTGG-3�; R158K/R160K, 5�-AATTCCAGTTTAGAGGCGGATTTGGTAAG-GGAAAGGGTCAGCCACCTCAGTAAC-3�, and 5�-TCGA-GTTACTGAGGTGGCTGACCCTTTCCCTTACCAAATC-CGCCTCTAAACTGG-3�. For GFP fusion of WT and R158K/R160K mutant constructs, the peGFP-N1 construct was digestedby XhoI/BamHI and inserted intoRPS10 using primers: 5�-CCG-CTCGAGGCCACCATGTTGATGCCTAAGAAG-3�, and 5�-CGGGATCCCCCTGAGGTGGCTGACCACG-3�. ForFLAG-tagged RPS10, a FLAG tag was added to the C terminus by PCRand subcloned into the SalI site of pcDNA3.1. Primers usedwere: 5�-CCGCTCGAGGCCACCATGTTGATGCCTA-AGAAG-3� and 5�-ACGCGTCGACTTACTTATCGTCGTC-ATCCTTGTAATCCTGAGGTGGCTGACC-3�. The FLAG-taggedRPS10-R158A/R160A plasmidwas constructed by pointmutagenesis in pcDNA3.1-RPS10-FLAG with the followingprimers: 5�-GTTTAGAGGCGGATTGGTGCCGGAGCAG-GTCAGCCACCTCAG-3� and 5�-CTGAGGTGGCTGA-CCTGCTCCGGCACCAAATCCGCCTCTAAAC-3�.PRMT5 cDNA was purchased from Open Biosystems. To

prepare the FLAG-tagged PRMT5, PRMT5was subcloned intopCMV-tag 2B (Stratagene) vector by PCR using primers: 5�-CGGGATCCATGGCGGCGATGGCGGTCG-3� and 5�-CCGCTCGAGGAGGCCAATGGTATATGAGC-3�; for theGFP fusion PRMT5 construct, the same PCR product wascloned into the BglII/SalI site of peGFP-C1. DsRed fusion B23andMyc-tagged B23 constructs were subcloned by PCR ampli-fication from pET21a-B23 (47). The primers 5�-GGAATTCA-CCATGGAAGATTCGATGGAC-3� and 5�-CGGGATCCCA-AAGAGACTTCCTCCACTG-3� were used for PCR andinserted into EcoRI/BamHI sites of pDsRed-N1 and pcDNA3.1Myc His(�) B. For GST fusion and FLAG-tagged B23, the B23was amplified by PCR using primers 5�-CGGGATCCATGGA-AGATTCGATGGAC-3� and 5�-CCGCTCGAGAAGAGAC-TTCCTCCACTG-3�. The PCR product was inserted into theBamHI/XhoI site of pGST-4T-1 and pCMV-tag 2B.

Purification of GST and GST-RPS10 Fusion Proteins

GST, GST-RPS10 wild type, and different mutants wereexpressed in E. coli BL21(DE3) and purified as described previ-ously (44). GST fusion proteinswere eluted from the beadswithelution buffer (10 mM Tris-HCl, 20 mM reduced glutathione,

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pH 8.0). The result of purification is shown in supple-mental Fig. S1.

RNA Interference Vector and siRNA-resistant Construction

RNA interference plasmids were prepared as previouslydescribed (48). For PRMT5 siRNA, 5�-GAGAAGATTCG-CAGGAACTC-3� and 5�-GCTGACCTCCCATCTAATC-3�were selected and designed as shRNAs in pNeoU6 vector.For RPS10, 5�-GAGCTGGCAGACAAGAAT-3� and 5�-GAG-TCATGGTGGCCAAGAA-3� were selected and namedsiRPS10-A and siRPS10-B. The oligonucleotides were annealedand cloned into RNAi-Ready pSIREN-DNR-DsRed (BD Bio-sciences). siRNA-resistant RPS10 constructs were generated bysite-directed mutagenesis of pcDNA3.1 RPS10-Myc using theQuikChange mutagenesis kit (Stratagene).

In Vitro Methylation Assay with GST Fusion Proteins

In vitro methylation reactions were performed in 40 �l ofmethylation buffer (25 mM Tris, 0.1 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride, 50 mM NaCl, pH 8.5) containing 3 �gof substrate and FLAG-PRMT5 immunopurified from trans-fected HEK293 cell extract. All methylation reactions were car-ried out in the presence of 0.42 �M [3H]S-adenosyl-L-methio-nine (GE Healthcare). The reaction was performed at 30 °C for1 h, separated on SDS-PAGE, treated with En3hanceTM(PerkinElmer Life Sciences), and exposed to film overnight.

Immunoprecipitation, Western Immunoblotting, and in VitroBinding Assay

Immunoprecipitation, Western immunoblotting, and invitro binding assay were performed as previously described(49, 50).

Nucleus/Cytosol Isolation

To isolation the cytosol and nuclear components, cells werewashed in ice-cold phosphate-buffered saline 24 h after trans-fection and collected by centrifugation in a microcentrifugetube. Buffer A (50 mM NaCl, 10 mM HEPES, pH 8.0, 500 mM

sucrose, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine,0.2% Triton X-100 and protease inhibitors) was added to thecell pellet and vortexed. The nuclei were spun out in a refriger-ated microcentrifuge at 5,000 � g for 2 min; and the superna-tant was kept as the cytoplasmic extract. The nuclear pellet waswashed with buffer B (50mMNaCl, 10 mMHEPES, pH 8.0, 25%glycerol, 0.1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermineand protease inhibitors). The supernatant was carefullyremoved and stored as the nuclear extract.

Cell Proliferation and Rescue Assay

24 h after transfection, 105 cells were seeded into each well of12-well plate. Medium was changed every 2 days throughoutthe course of the experiments. For each sample, three fieldsfrom each well in three duplicated wells were selected ran-domly, counted under a fluorescencemicroscope, and analyzedseparately every 2 days.

Protein Half-life Assay and Ubiquitination Assay

To measure the half-life of proteins, HEK293 cells weretransfected with RPS10-FLAG or its mutant cDNAs. 24 h later,

cells were treated with 100 �g/ml of cycloheximide (CHX)together with or without 10 �M MG132 for 0, 2, 4, 6, and 8 h.The cells were harvested, and cell lysates were analyzed byimmunoblotting with anti-FLAG and anti-tubulin antibod-ies. For pulse-chase experiments, 48 h after transfection,medium was replaced with methionine-cysteine-free me-dium for 40 min. The cells were then incubated in freshmethionine-cysteine/fetal bovine serum-free Dulbecco’smodified Eagle’s medium containing 10�Ci/ml of [35S]methi-onine (PerkinElmer Life Sciences) for 1 h. Complete medium(containing methionine, cysteine, and fetal bovine serum) wasadded and incubated for the desired chase times. Total celllysate was immunoprecipitated with FLAG beads, resolved by10% SDS-PAGE, and visualized by autoradiography. The ubiq-uitination assay was performed as described previously (49).

Measurement of Protein Synthesis

Cells were incubated inmethionine-cysteine-freeDulbecco’smodified Eagle’s medium for 30 min before labeling. In vivometabolic labelingwas performed by adding Trans35S-label at afinal concentration of 50 mCi/ml containing labeled L-methio-nine. After 90 min, the cells were washed with ice-cold phos-phate-buffered saline and precipitatedwith 10% trichloroaceticacid, washed with ice-cold acetone, and vacuum-dried. Totalprotein content was measured using the Bradford assay, andproteins were separated by SDS-PAGE and exposed to film.

Image Acquisition and Analyses

24 h after transfection, cells were fixedwith 2% formaldehydesolution in phosphate-buffered saline. Confocal images werecapturedonaLeicaTCSSP5confocalmicroscope.Immunofluo-rescent images were scanned with a 1042 � 1024 frame size,4� zoom, and �1.0 �m optical thickness. Detector gain andamplifier offset were adjusted to ensure all signals wereappropriately displayed within the linear range. Exposuretime was set so that the brightest intensity reached 80% ofthe saturation intensity.

Polysome Isolation and Polysome Profiling

Polysome Isolation—48 h after transfection, cells were treatedwith 100 �g/ml of CHX for 15 min, and then washed with ice-cold phosphate-buffered saline (containing 100 �g/ml ofCHX). The cell extract was prepared using 500 �l of polysomelysis buffer (1.0% Triton X-100, 20mMTris, pH 7.5, 50mMKCl,10 mM MgCl2, 1 mM dithiothreitol, 0.5 unit/�l of RNase inhib-itor, 100 �g/ml of CHX, 0.2 mg/ml of heparin and proteaseinhibitors). Following a 10-min incubation on ice, cellulardebris was discarded after centrifugation at 14,000 � g for 15min at 4 °C. The supernatants were centrifuged for 1 h at40,000 � g at 4 °C in a Beckman SW41Ti rotor. Pellets wereresuspended in polysome buffer and separated by SDS-PAGEand analyzed by Western blotting. The ratio of RPS10 in poly-some/total cell lysate was analyzed with the Image J 1.36b soft-ware (rsb.info.nih.gov/ij).Polysome Profiling—Cells were treated with CHX and lysed

as described above. The supernatant was layered over a 5–45%(w/v) sucrose density gradient in ribosome lysis buffer andultracentrifuged in a swinging bucket MLA-55 rotor at

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38,000 � g for 3 h at 4 °C. Total layers containing the ribosome80 S, 60 S large subunit and 40 S small were collected. Thegradients were next fractionated by upward displacement usinga Biologic Duo-flow (Bio-Rad) apparatus connected to a UVdetector for continuous absorbance measurements at 254 nm.

RESULTS

RPS10 Is a Substrate of PRMT5—During our study of theleucine-rich repeat kinase 2 (LRRK2)-associated complex, wepurified a group of endogenous LRRK2-interacting proteins.3Within this group, we found several proteins that belong to apreviously reported complex,methylosome, including PRMT5,MEP50 (methylosome protein 50, a scaffold for methylosome)(31), and several substrates for PRMT5 such as histone 2A andhistone 4. This suggests that the LRRK2-associated complexmay play a role in proteinmethylation. Because the glycine- andarginine-richmotif (GARmotif) is a conservedmethylation sitefor a subset of arginine methyltransferases, we searched forother LRRK2-associated proteins that contain the GAR motifand found RPS10.RPS10 is a member of the ribosome 40 S subunit complex.

We hypothesized that RPS10 is a substrate of PRMT5 based onthe following evidence. First, mass spectrometry results for 40 Shuman ribosome indicate that RPS10 is methylation modified(32); second, a similar mass spectrometry analysis of rat brainextract uncovered an RPS10 C-terminal peptide with a 56-Damass larger than predicted (33); third, 87.1% of anti-Sm sera(which recognizes sDMA in Sm) from patients with systemiclupus erythematosus, has antibody activity against RPS10 (34).Finally, Y12, a specific symmetric dimethylation antibody, rec-ognizes the sDMA of Sm and cross-reacts with RPS10 (34).To test our hypothesis that RPS10 is a novel substrate of

PRMT5, we investigated first whether RPS10 and PRMT5interact with each other using reciprocal immunoprecipitationassay. In transfected cells, where different tagged PRMT5 andRPS10 proteins were co-expressed and subsequently immuno-precipitated, a specific complex between RPS10 and PRMT5was detected (Fig. 1,A andB). To evaluate whether endogenousPRMT5 and RPS10 interact, lysates of HEK293 cells were sub-jected to co-immunoprecipitation and Western blotting withPRMT5 and RPS10 antisera. Analysis of the immunoprecipi-tates revealed clear interactions between the endogenous pro-teins (Fig. 1C). To further characterize the interaction betweenPRMT5 and RPS10, we incubated purified GST andGST-fusedRPS10 with FLAG-PRMT5 purified from HEK293 cell lysates.Interactions between GST-RPS10 and FLAG-PRMT5 wereobserved (Fig. 1D).After recognizing that RPS10 interacts with PRMT5 under

physiological conditions, we next examined whether RPS10 is asubstrate of PRMT5. By incubating immunopurified FLAG-PRMT5 with GST-RPS10 and [methyl-3H]S-adenosylmethi-onine, we found that PRMT5 can methylate GST-RPS10 butnot GST (Fig. 2B). RPS10 contains several potential glycine-arginine-rich (GAR) motifs (Arg-Gly-Gly-Phe (RGGF) andArg-Gly (RG)) at its C-terminal end (Fig. 2A). To determine

whether this region in RPS10 is the methylation site or not, weconstructed a C-terminal GARmotif deletion mutant (�GAR).The removal of GAR motifs resulted in the loss of methylationmodification by PRMT5 (Fig. 2B). This indicates that PRMT5methylates RPS10 within its C-terminal GAR motifs.There are three arginine residues in the GAR motifs at the

RPS10 C terminus, namely Arg153, Arg158, and Arg160. Massspectrometry results show there is a 56-Da increase in themolecular mass of RPS10 (33), suggesting the existence of twodimethylated arginines. To determine the exact location of themethylation sites, we mutated the potential amino acids intolysine residues, either alone or in combination. The resultingR158K and R160K mutants of RPS10 showed weaker methyla-tion by PRMT5 and methylation of the R158K/R160K mutantwas totally lost (Fig. 2C). Arg158 and Arg160 of RPS10 are there-fore likely to be the sites of methylation by PRMT5.To investigate whether PRMT5 is required for the methyla-

tion of RPS10 and whether Arg158 and Arg160 of RPS10 are thearginine methylation sites in vivo, we used a symmetric di-methylarginine-specific antibody, SYM11, to detect the meth-ylated arginine residues in immunopurified RPS10 and RPS10-R158K/R160K proteins. As shown in Fig. 2D, the SYM11antibody clearly detected methylation inWTRPS10, but not inRPS10 (R158K/R160K) immunopurified from transfected 293cells. We then knocked down the expression of PRMT5 inU2OS cells with different shRNA and found that the levelsof methylation of endogenous RPS10 reduced significantly

3 J. Ren, Y. Wang, Y. Liang, Y. Zhang, S. Bao, and Z. Xu, manuscript inpreparation.

FIGURE 1. RPS10 interacts with PRMT5 both in vivo and in vitro. A andB, RPS10 interacts with PRMT5 in vivo. HEK293 cells were transfected with anexpression vector encoding tagged PRMT5 and RPS10 as indicated. 24 h later,aliquots of cell lysates were saved for analysis of expressed tagged proteins.The remaining portions of the lysates were immunoprecipitated with anti-FLAG for FLAG-PRMT5, and immunocomplexes were analyzed for Myc-RPS10(A). In reciprocal co-expression/immunoprecipitation (IP) experiments, celllysates were subjected to immunoprecipitation with anti-Myc antibody andimmunocomplexes were probed for the presence of GFP-PRMT5 (B).C, endogenous PRMT5 and RPS10 interact with each other. HEK293 celllysates were subjected to immunoprecipitation with anti-RPS10 or IgG anti-bodies as indicated. The immunocomplexes were probed separately forPRMT5 and RPS10. D, PRMT5 interacts with RPS10 in vitro. GST and GST-RPS10fusion protein for binding assays were expressed, purified, and incubatedwith FLAG-PRMT5 protein purified from HEK293 cell lysates with FLAG beads.GST, GST-RPS10, and FLAG-PRMT5 were detected with GST and FLAG antiseraseparately.

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(Fig. 2E). Taken together, these results indicate that PRMT5 canmethylate RPS10 at Arg158 and Arg160 both in vitro and in vivo.Methylation of RPS10 Is Required for Normal Cell Prolifera-

tion—Because a group of ribosomal proteins have been re-ported to regulate cell cycle and cell proliferation (35, 36), weinvestigated the physiological role of RPS10 in cell prolifera-tion.We generated two different shRNA-expressing constructsto knock down the expression of endogenous RPS10 in Bel7402cells. As shown in Fig. 3,A–C, endogenous RPS10 protein levelswere reduced significantly 2 days after transfection, and cellstransfected with siRPS10 constructs grew much slower thancells transfected with control siRNA. By day 5, cells transfectedwith control siRNA, but not those transfected with siRPS10

constructs, formed colonies (Fig. 3A). Similar results wereobtained for the other cell lines, including U2OS and HEK293(data not shown). This indicates that RPS10 is required for sus-tained cell proliferation.To examine whether methylation of RPS10 plays a role in

proliferation, we compared the rescue effects ofWTRPS10 andthe RPS10 methylation mutant in RPS10 knock-down cells.First, we designed both WT and methylation mutant siRNA-

FIGURE 2. PRMT5 methylates RPS10 both in vitro and in vivo. A, alignmentof the conserved RG-rich motif from human (Homo sapiens), mouse (Mus mus-culus), Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharo-myces pombe. Highly conserved residues are highlighted in black; weakly con-served residues are highlighted in gray. B, PRMT5 methylates the C-terminalof RPS10 in vitro. Methylation of GST, GST-RPS10, or the GAR motif deletionmutant (�GAR) of RPS10 by immunopurified FLAG-PRMT5 was performed asdescribed under “Experimental Procedures” and visualized by SDS-PAGE sep-aration followed by Coomassie Brilliant Blue staining (left) and autoradiogra-phy (right). C, Arg158 and Arg160 in RPS10 are the residues methylated byPRMT5 in vitro. In vitro methylation assays of GST-RPS10, GST-RPS10 �GAR,and arginine single or double mutants of RPS10 (GST-RPS10-R158K, -R160K,or -R158K/R160K) by immunopurified FLAG-PRMT5 were performed as in B.D, PRMT5 methylates RPS10 in vivo. HEK293 cells were transfected with FLAG-tagged RPS10 or RPS10-R158K/R160K together with PRMT5. 24 h later, celllysates were immunoprecipitated with anti-FLAG antibody and probed withSYM11 to detect symmetric dimethylarginine in RPS10. The blot wasre-probed with anti-FLAG antibody for RPS10 or RPS10-R158K/R160K.E, PRMT5 is required for the methylation of RPS10 in vivo. HEK293T cells weretransfected with FLAG-RPS10 together with shRNA expressing vectors target-ing either luciferase or PRMT5 in a ratio of 1:5. 48 h after transfection, celllysates were analyzed by immunoblotting with anti-PRMT5 antiserum (leftpanels, GAPDH used as a loading control). Immunoprecipitates (IP) obtainedusing the FLAG antibody from cell lysates were analyzed with anti-FLAG anti-body for RPS10 and SYM11 antibody for symmetric dimethylarginine inRPS10 (right panels).

FIGURE 3. Methylation of RPS10 by PRMT5 is required for normal cellproliferation. A and B, RPS10 is required for cell proliferation. pSIREN-DNR-DsRed.siLuciferase, pSIREN-DNR-DsRed.siRPS10 A, and pSIREN-DNR-DsRed.siRPS10 B (labeled as Control, siRPS10 A, and siRPS10 B, respectively) were co-transfected into Bel7402 cells. 24 h later, cells were split into 6-well plates andallowed to grow for 5 more days. A, transfected cells are shown on days 1 (top)and 5 (bottom) under a fluorescence microscope (�10). B, the number oftransfected cells was counted daily under a fluorescence microscope. Theexperiment was repeated three times and values represent average numbersof transfected cells and are means from duplicated cultures � S.E. C, RPS10shRNAs can down-regulate the expression of endogenous RPS10. Bel7402cells were transfected as in A. 36 h later, RPS10 expression was determined byWestern blotting using RPS10 antiserum. The blot was re-probed with �-tu-bulin as a loading control. D and E, WT RPS10 restored cell proliferation betterthan the methylation mutant in the RPS10 knock-down cells. Myc-tagged WTand methylation mutant siRNA-resistant RPS10 (SR-WT and SR-R158K/R160K)were generated by changing three nucleotides, as underlined, without alter-ing the amino acid sequences (D, lower panel). Bel7402 cells were co-trans-fected with siRPS10 together with the control, SR-WT, or SR-R158K/R160K.36 h later, cells were harvested, and half of the cells were split into 6-wellplates for rescue experiments (E). D, expression of endogenous and exoge-nous RPS10 was determined as in C. The number of transfected cells wascounted every 2 days for 6 days. The experiment was repeated four timesand values were expressed as the means � S.E. (day 4, p � 0.05; day 6, p �0.01) (E).

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resistant RPS10 (SR-RPS10) by changing three nucleotides inRPS10 without altering the amino acid sequence (Fig. 3D).Bel7402 cells were co-transfected with siRPS10 together witheither the siRNA-resistant WT or methylation mutant ofRPS10. As shown in Fig. 3E, expression of theWT SR-RPS10 inthe RPS10 knock-down cells restored cell proliferation moreeffectively than the methylation mutant SR-RPS10. Westernblotting of total cell lysates revealed that RPS10 protein levelswere down-regulated in siRPS10-transfected cells and expres-sion of siRNA-resistant RPS10s (WT or methylation mutant)restored the total RPS10 levels similar to those of the control(Fig. 3D). Thus, we speculate that methylation of RPS10 plays arole in the appropriate cell proliferation. To further supportthis conclusion, we were able to establish permanent cell lineswith SR-RPS10, but not with SR-RPS10-R158K/R160K, in cellsexpressing RPS10 shRNA (supplemental Fig. S2 and data notshown).Methylation of RPS10 Plays a Role in the Efficient Assembly of

RPS10 into Ribosomes—Because methylation of RPS10 is im-portant for cell proliferation, we went on to investigate theunderlying mechanism by examining the assembly of RPS10into ribosomes. WT RPS10, or the RPS10 methylation mutant,was transfected into HEK293 cells and ribosomes were isolatedby ultracentrifugation. As shown in Fig. 4A, translating poly-somes were pooled in the ultracentrifugation pellet. Endoge-nous RPS10 was present, as expected, in all the ribosomal frac-tions including the total cell lysate and supernatant andpolysome fractions, whereas GFP and GAPDH were detectedonly in total cell lysates and supernatant fractions. The FLAG-tagged WT RPS10 protein was present in the free cytoplasmicfractions as well as polysomes indicating that it can be assem-bled into ribosomes properly.To determine whether the RPS10 methylation mutant was

assembled into ribosomes as efficiently as WT RPS10, we ana-lyzed the fraction of exogenous RPS10 associated with poly-somes using ImageJ software. The percentage of polysome-as-sociated exogenous RPS10 was calculated as RPS10-FLAG inpolysomes/endogenous RPS10 in polysomes and normalizedwith RPS10-FLAG in total cell lysates/endogenous RPS10 intotal cell lysates (Fig. 4B). The fraction of the RPS10-R158K/R160K mutant associated with ribosomes was less than that ofWT RPS10 (Fig. 4B), indicating that the RPS10 methylationmutant was assembled into ribosomes less efficiently.Rmt3, the homolog of PRMT3 in humans, has been charac-

terized as a methyltransferase for ribosomal protein Rps2 (21,42). RMT3-null cells produce nonmethylated Rps2 and showderegulation of the 40 S:60 S ribosomal subunit ratio (21).Because the RPS10 methylation mutant is less efficient inassembling into ribosomes, we analyzed ribosome profiles insucrose gradients, including the levels of free 40 S and 60 Sribosomal subunits and 80 S monosomes, to determinewhether ribosome biosynthesis is affected. As was the casewhen yeast Prmt3 was disrupted, expression of RPS10-R158K/R160K in cells when endogenous RPS10 was knocked down byshRNA led to perturbation of the 40 S:60 S ribosomal subunitratio. The A254 60 S:40 S ratio for cells expressing wild-typeRPS10 was 2.01; the A254 ratio from three independent experi-ments increased by about 19.5% to 2.40 in cells expressing

mutant RPS10 (Fig. 4C, arrow in peak). This suggests that themethylation mutation of RPS10 may lead to misregulation ofthe 40 S:60 S ribosomal subunit ratio.To examine whether the ribosomal subunit imbalance seen

in cells expressing mutant RPS10 affects global protein synthe-sis in cells, total protein was labeled with [35S]methionine andseparated on an SDS-PAGE gel. As shown in Fig. 4,D and E, thelevels of most newly synthesized proteins from cells expressingmutant RPS10 were lower than those from cells expressingWT

FIGURE 4. Methylation of RPS10 plays a role in the efficient assembly ofRPS10 into ribosomes and protein synthesis. A, cytoplasmic polysomeswere isolated by ultracentrifugation as described under “Experimental Proce-dures.” The polysome fraction was extracted from HEK293 cells transfectedwith FLAG-tagged WT RPS10 or R158K/R160K mutants together with pCMS.EGFP. Collected fractions were analyzed by Western blotting using antibodiesspecific for RPS10, GFP, and GAPDH. T, S, and P represent total cell lysates,supernatant, and isolated polysomes, respectively. B, quantitative analysis ofthe fraction of exogenous RPS10 associated with polysomes was calculatedas RPS10-FLAG in polysomes/endogenous RPS10 in polysomes and normal-ized with RPS10-FLAG in total cell lysates/endogenous RPS10 in total celllysates by ImageJ software. Values are three independent experiments,mean � S.E. (p � 0.05). C, polysome profile. 48 h after transfection, totalextracts from HEK293T cells transfected with RPS10 shRNA together witheither SR-RPS10-Myc or methylation mutation RPS10 were subjected to5– 45% linear sucrose density gradient sedimentation by ultracentrifugation.The linear peak represents a continuous measurement of the absorbance at254 nm. D, RPS10 methylation mutation leads to a decreased protein synthe-sis rate. Cells were transfected as in C. 48 h after transfection, the medium wasexchanged to a Met-free medium supplemented with [35S]Met. Cells wereharvested 1 h after labeling and then subjected to SDS-PAGE analysis. TheSDS-PAGE gel was stained with Coomassie Blue (left, lane 1, WT SR-RPS10/RPS10 shRNA; lane 2, SR-RPS10 methylation mutant/RPS10 shRNA), dried, andexposed to x-ray film for 35S-labeled proteins (right, lanes 3 and 4 correspondto lanes 1 and 2). E, quantitative analysis of total protein synthesis in D calcu-lated as 35S radioactivity/total protein (Coomassie Blue staining) usingImageJ software. Values are three independent experiments mean � S.E.(*, p � 0.01).

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RPS10 (right panel, comparing those bands labeled by arrows inlane 4 with lane 3), however, there was not much difference inthe total protein levels (left panel, compare lanes 1 and 2). Wenoticed that only one band corresponding to the size of over-expressed DsRed showed no apparent change (dashed arrow).Our data suggests that biogenesis of new proteins is slower incells expressingmutant RPS10 comparedwith those expressingWT RPS10.The RPS10 Methylation Mutant Is Unstable Compared with

WT RPS10—Ribosomal proteins are expressed at high levelsand different unassembled ribosomal proteins have beenshown to be degraded in the nucleoplasm (37).We noticed thatexogenously expressed RPS10-R158K/R160K was consis-tently present at lower levels than WT RPS10 (supplementalFigs. S2 and 4C). This led us to investigate the relationshipbetweenmethylation of RPS10 and its stability. WT RPS10 anddifferent amounts of RPS10-R158K/R160K were co-trans-fected with EGFP expression vector into 293 cells. As shown inFig. 5A, the expression level of WT RPS10 was much higherthan that of the mutant. The expression of the mutant waslower even though 3 times more of the mutant was transfectedand with a higher transfection efficiency as indicated by EGFP(Fig. 5A). To test whether rapid degradation could account forthe low expression level of the mutant RPS10 protein, CHX (aninhibitor of de novo protein synthesis) pulse-chase experimentswere performed. As shown in Fig. 5B, levels of RPS10 declinedin the presence of CHX. However, we found that the RPS10-R158K/R160Kmutant wasmuchmore unstable (the half-life ofRPS10-R158K/R160K was �5 h, whereas that of WT RPS10was around 8 h as analyzed with ImageJ software). As CHXtreatment has been reported to affect ribosome assembly andstability of subunits, we used [35S]methionine pulse-chase anal-ysis of quantitative immunoprecipitations of WT RPS10 andthe RPS10-R158K/R160Kmutant with PRMT5 as a control. Asshown in Fig. 5D, the RPS10-R158K/R160Kmutant was appar-ently less stable than WT RPS10.Many ribosomal proteins are degraded in a proteasome-de-

pendent manner (37). To determine whether RPS10 is alsodegraded through the proteasomal pathway, we analyzed theeffect of the proteasome inhibitor, MG132, on the degradationof RPS10. As shown in Fig. 5C, both WT RPS10 and RPS10-R158K/R160K accumulated rapidly in the presence of MG132.To further examine whether or not RPS10 expression is subjectto regulation by ubiquitination,WT RPS10 and its methylationmutant were co-transfected into HEK293 cells together withMyc-tagged ubiquitin. RPS10 was immunoprecipitated and theubiquitination level was analyzed. We detected a high molecu-lar weight smear above RPS10 with its intensity significantlyenhanced by MG132 treatment (Fig. 5E, lane 2 versus lane 5).Ubiquitination of the RPS10 methylation mutant was muchmore apparent than forWTRPS10, especially in the presence ofMG132 (Fig. 5E, lane 5 versus lane 6). Taken together, ourresults indicate that theRPS10methylationmutant is less stableand is prone to degradation by the proteasomal pathway.Methylation of RPS10 Is Required for Its Concentration in the

GC Region of Nucleoli—Ribosome biogenesis takes place in thenucleolus, a specialized compartment within the nucleus. Eachnucleolus contains three morphologically distinct components

FIGURE 5. The RPS10 methylation mutant is less stable than WT RPS10.A, expression of RPS10 methylation mutant is much lower than that of WTRPS10. HEK293 cells were transfected with FLAG-tagged RPS10, or differ-ent amounts of RPS10-R158K/R160K, together with 1 �g of pCMS.EGFP.36 h later, RPS10 expression was determined by Western blotting usinganti-FLAG antibody. The blot was re-probed with anti-GFP antibody as atransfection efficiency control. B and C, RPS10 methylation mutant is moreunstable and can be stabilized by the proteasome inhibitor. HEK293 cellswere transfected with FLAG-tagged RPS10 or RPS10-R158K/R160K. 36 hlater, cells were treated with either 100 �M cycloheximide (CHX) or 10 �M

MG132 and cells were collected at different time points as indicated.RPS10 expression levels were determined by Western blotting usingRPS10 antiserum. The blot was re-probed with �-tubulin as a loading con-trol. D, HEK293 cells were transfected with either WT RPS10-FLAG orRPS10-R158K/R160K-FLAG together with FLAG-PRMT5. Pulse-chase exper-iments were done by labeling with [35S]methionine for 1 h and a subse-quent chase with medium containing non-radioactive methionine for theindicated times before cells were lysed. WT and mutant RPS10 and PRMT5proteins were immunoprecipitated with anti-FLAG beads. 35S-LabeledRPS10-FLAG was analyzed with a FLA-3000 phosphorimager, and FLAG-PRMT5 was used as a control. E, the RPS10 methylation mutant is prone todegradation by the proteasomal pathway. HEK293 cells were co-trans-fected with a control vector (lanes 1 and 4), RPS10-FLAG (lanes 2 and 3), orRPS10-R158K/R160K (lanes 5 and 6) together with Myc-tagged ubiquitin.36 h after transfection, the cells were treated with MG132 (lanes 4 – 6) asindicated. FLAG-tagged RPS10 was immunoprecipitated using anti-FLAGbeads, subjected to SDS-PAGE, and immunoblot (IB) analysis to detect theubiquitylated RPS10 or RPS10-R158K/R160K with anti-Myc antiserum. Theblot was reprobed with anti-FLAG antiserum to detect RPS10 andRPS10-R158R/K160K.

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that reflect the process of ribosomeproduction. Synthesis, modifica-tion, and initial cleavage of pre-rRNA take place within the fibrillarcomponents and the surroundingdense fibrillar component, whereaslater processing steps and assemblyof ribosomes are performed in theGC region (38).We determined the localization

of RPS10 in the Bel7402 permanentcell line (which stably expressesRPS10-Myc) by immunostainingand expected it to localize in thenucleolus as other ribosomal pro-teins (39). RPS10-Myc was localizedin nuclei where it appeared to accu-mulate primarily in structures cor-responding to nucleoli (supplementalFig. S3). A weaker and diffuse cyto-plasmic staining was also noticed.The ring-shaped subcellular local-ization of RPS10 in the stable cellline corresponds to that of NPM/B23, a well known marker for theGC region of nucleoli, and was verysimilar to that of RPS10 fused toGFP in transiently transfected cells(supplemental Fig. S4 and data notshown). This led us to test whetherRPS10 is concentrated in the GCregion of nucleoli by co-transfectingB23 with either RPS10 or RPS10-R158K/R160K into U2OS cells. Wefound that 57% of WT RPS10mainly co-localized with B23 sug-gesting that RPS10 was concen-trated in the GC region (Fig. 6, Aand E). To our surprise, co-localiza-tion of RPS10-R158K/R160K withB23 was not found in any cells.Instead, its signal intensified insidenucleolar areas other than the GCregion (Fig. 6, B and E). To furtherdefine the location of RPS10, WTRPS10 and RPS10-R158K/R160Kwere co-transfected with Fibrillarinseparately. As shown in Fig. 6,C andD, mutant RPS10, instead of WTRPS10, co-localizedwell with Fibril-larin. These results indicate thatRPS10 is enriched in the GC regionof nucleoli and that methylation ofRPS10 is required for its concentra-tion in the GC region of nucleoli.As methylation of RPS10 is re-

quired for its concentration in theGC region of nucleoli and plays a

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role in the assembly of RPS10 into ribosomes, we went on toinvestigate whether the distribution of RPS10 in the whole cell,especially its export to the cytosol, is affected by methylation.WT RPS10 and RPS10-R158K/R160K were transfected intoHEK293 cells. Nuclear and cytoplasmic fractions were sepa-rated and analyzed 48h later. Using LaminB as amarker, Fig. 6Fshows thatmore RPS10-R158K/R160Kmutant is present in thenuclear fraction. Thus, our results suggest that methylation ofRPS10 plays a role in its location in the GC region of nucleoliand may also play a role in its transport from the nucleus to thecytosol.The RPS10 Methylation Mutant Shows Decreased Binding to

B23—B23/NPM, also known as Numatrin1, plays a key role inthe processing and/or assembly of ribosomes (40). The chaper-one properties of B23might be a key to this process by prevent-ing the aggregation of proteins and selectively storing a subsetof ribosomal proteins to facilitate ribosome biogenesis (41, 45).The co-localization of RPS10 with B23 led us to determinewhether RPS10 interacts with B23. Reciprocal co-immunopre-cipitation assays using lysates fromcells transfectedwith taggedB23 and/or RPS10 were carried out and confirmed the specificinteraction between RPS10 and B23 (Fig. 7, A and B). To eval-uate whether endogenous RPS10 and B23 interact, lysates fromHEK293 cells were subjected to co-immunoprecipitation andWestern blotting with RPS10 and B23 antisera. Analysis ofthe immunoprecipitates revealed clear interaction between theendogenous proteins (Fig. 7C). To determine whether theinteraction between B23 and RPS10 is direct or indirect, GSTpull-down assays using B23 and RPS10 expressed and purifiedfrom E. coliwere employed. As shown in Fig. 7D, GST-B23, butnot GST, could pull-down His-tagged RPS10 suggesting thatRPS10 interacts with B23 directly.We showed above that most of the WT RPS10 co-localizes

with B23 in nucleoli and that the RPS10 methylation mutantfails to do so. We therefore investigated whether there are dif-ferences in the interaction between B23 and theWTandRPS10methylationmutant using co-immunoprecipitation assays. It isinteresting to note that interactions between RPS10-R158K/R160K and either overexpressed or endogenous B23 wereweaker than those between WT RPS10 and either overex-pressed or endogenous B23 (Fig. 7, E and F). Taken together,these results indicate that RPS10 is a novel B23-binding proteinand the methylation mutation of RPS10 leads to decreasedbinding to B23 and it thus may explain why the RPS10 methyl-ation mutant failed to co-localize with B23/NPM in the GCregion of nucleoli.

DISCUSSION

RPS10 Is a Novel Substrate for PRMT5—Arginine methyla-tion of ribosomal proteins is common in prokaryotes andeukaryotes. Although several methylated ribosomal proteins

FIGURE 6. Methylation of RPS10 is required for its concentration in the GC region of nucleoli. A and B, difference in the subnucleolar localization of WTRPS10 and RPS10 methylation mutant. RPS10-GFP (A) and RPS10-R158K/R160K-GFP (B) were co-transfected with B23-DsRed into U2OS cells and fixed 24 h later.RPS10 (green) and B23 (red) were observed under a confocal microscope. High magnifications of the indicated areas (squares) are shown at the bottom. Bars,5 �m. The graphs correspond to intensities in arbitrary units of green and red labeling for each pixel of the line drawn through the axis in A and B, respectively.C and D, RPS10-GFP (C), RPS10-R158K/R160K-GFP (D), and Fibrillarin-DsRed were transfected and analyzed as in A and B. E, different distributions of WT RPS10and methylation mutant in nucleoli. GFP-tagged RPS10 and RPS10-R158K/R160K were co-transfected either with B23-DsRed or Fibrillarin-DsRed into U2OScells, fixed 24 h later, and observed as in A and C. RPS10 was classified as either GC region localization when co-localized with B23, or dense fibrillar component(DCF)/fibrillar component (FC) when co-localized with Fibrillarin. 100 cells were analyzed for each category. F, more RPS10-R158K/R160K tends to remain in thenucleus. HEK293 cells were transfected with FLAG-tagged WT or methylation mutant RPS10. 24 h after transfection, cytoplasmic and nuclear components werefractionated. WT and mutant forms of RPS10 in total lysate (T), cytosol (C), and nuclear (N) were analyzed by Western blot with anti-FLAG antibodies. Lamin Band GAPDH were used as the marker for nuclear and cytoplasmic fractions, respectively.

FIGURE 7. Decreased binding of RPS10 methylation mutant to B23. A andB, RPS10 interacts with B23 in vivo. HEK293 cells were transfected with FLAGor Myc-tagged RPS10 and B23 as indicated. 24 h later, cell lysates were immu-noprecipitated with anti-FLAG beads and the immunocomplexes were ana-lyzed for either Myc-tagged RPS10 or B23. C, endogenous B23 and RPS10interact with each other. Anti-RPS10 mouse serum was used to immunopre-cipitate (IP) B23 from HEK293 cell extracts with mouse IgG as a control. Thecell lysates (lower panels) and immunocomplexes (upper panels) were probedseparately for endogenous B23 and RPS10. D, RPS10 interacts with B23 invitro. GST, GST-B23 fusion protein, and His-RPS10 for binding assays wereexpressed, purified from E. coli, and incubated with each other. GST, GST-B23,and His-RPS10 were detected with GST and His antisera separately.E, decreased binding of RPS10 methylation mutant to B23. HEK293 cells weretransfected with FLAG-tagged B23 together with either Myc-tagged RPS10 orRPS10-R158R/K160K. The amount of RPS10-R158K/R160K used for transfec-tion was doubled so that its expression level is similar to that of WT RPS10.24 h later, cell lysates were analyzed for the expression of tagged proteins(lower panel). The remaining portions of the lysates were immunoprecipi-tated with anti-FLAG antibody, and the immunocomplexes were analyzed forRPS10 or RPS10-R158K/R160K with anti-Myc antibody. F, decreased bindingof RPS10 methylation mutant to endogenous B23. HEK293 cells with eitherMyc-tagged RPS10 or RPS10-R158K/R160K. The amount of RPS10-R158K/R160K used was tripled. Cell lysates were immunoprecipitated with anti-B23antibody, and the immunocomplexes were analyzed for RPS10 or RPS10-R158K/R160K with anti-Myc antibody and B23 with anti-B23 antibody.

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have previously been identified, the biological significance ofthis methylation is still poorly understood (43).In this study, we found that RPS10 interacts with PRMT5

both in vivo and in vitro and it is a novel substrate for PRMT5.A knock-down experiment using shRNA indicated thatPRMT5 played a role in the methylation of RPS10. Because thedecrease of RPS10 methylation levels is not very significantdespite efficient knockdown of PRMT5 (Fig. 2E), it is likely thatRPS10 can be methylated by other PRMT family members aswell. The methylation sites in RPS10 were identified as Arg158and Arg160.Methylation of RPS10 Plays a Role in the Biogenesis of Ribo-

somes and Protein Synthesis—Ribosomal proteins are assem-bled in the nucleolus, especially in the GC region, to build theribosome (38). They are then exported into the cytoplasm asribosomal subunits. The role of ribosomal protein post-tran-scriptional modification in this process is poorly understood.We found thatWTRPS10 is enriched in theGC region of the

nucleolus and that the methylation mutant failed to do so. Thiselucidates, at least in part, why the methylation mutant ofRPS10 was not assembled into ribosomes as efficiently as WTRPS10. Methylation of RPS10 is likely to promote its interac-tion with B23 and its enrichment in the GC region. However, itis also possible that methylation of RPS10 induces the localiza-tion of RPS10 to the GC region and facilitates its interactionwith B23 there.It is interesting to note thatmethylation of RPS10 plays a role

in the assembly of RPS10 into ribosomes. It is also important forribosome biogenesis because mutation of RPS10 leads to mis-regulation of the 40 S:60 S ribosomal subunit ratio and subop-timal protein synthesis. Thiswould explainwhy knocking downRPS10 expression with shRNA almost completely halted cellproliferation, which could then be rescued byWTRPS10 but toa lesser extent by the RPS10 methylation mutant.It has been shown previously that unassembled ribosomal

proteins are degraded quickly, whereas cytoplasmic pools ofribosome-associated ribosomal proteins are relatively stable(37). This may account for why the RPS10methylationmutant,which was not assembled into ribosomes and exported to thecytoplasm efficiently, was less stable than WT RPS10 anddegraded faster in a proteasome-dependent manner.PRMT5, Methylation of RPS10, and Tumorigenesis—Both

PRMT5 and ribosomal activity have been shown to be closelyassociated with cell proliferation and tumorigenesis (1, 22, 23).Like PRMT5, B23 is involved in many forms of tumors (40, 41).Our findings support a model in which PRMT5 may regulatecell growth via methylation of RPS10. Methylation of RPS10 byPRMT5 is likely to play a role in its interactionwithB23, proteinstability, localization to the GC region of the nucleolus, andefficient assembly into ribosomes.It will be interesting to examine in the future whether

PRMT5 controls cell proliferation through the regulation ofribosome assembly and protein synthesis. Accumulating evi-dence suggests that some proteins can be methylated by bothtype I and type II PRMTs. Thus, aDMAand sDMAmethylationof the same proteinmay lead to different and possibly opposingbiological consequences. It will therefore be intriguing to inves-tigate whether other PRMT family members, in addition to

PRMT5, play a role in themethylation, localization, and assem-bly of different ribosomal proteins and the regulation of cellproliferation.PRMT5, Methylation of RPS10, and Systemic Lupus Erythe-

matosus—Systemic lupus erythematosus (SLE) is characterizedby the presence of circulating autoantibodies to cellular constit-uents. Among these, anti-Sm antibodies are found inmore than30%of patientswith SLE and are used as a diagnosticmarker forSLE (46). Our results indicate that RPS10 is dimethylated byPRMT5, and others have reported that anti-RPS10 antibodieswere found in the sera of 11% of total SLE patients, and in 31%of active SLE patients. A high frequency of antibody activityagainst RPS10 (87.1%) has been found in anti-Sm sera from SLEpatients. These patients tend to be in the active stage of SLE(34). More interestingly, sDMA of RGmotifs in Sm and RPS10are the major sites recognized. This raises a further question:what is the exact role of symmetrically dimethylated Sm andRPS10 in SLE? PRMT5 has previously been shown to producesDMAs in the RG motifs of Sm proteins (17) and in RPS10 inthis study. In addition to studying the relationship betweenPRMT5 and tumorigenesis, it will be intriguing to study in thefuture whether PRMT5 is also associated with SLE.

Acknowledgments—We thank Drs. L. Greene, J. Zhang, C. Yang, andX.Huang for comments and suggestions during the preparation of thismanuscript.

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PRMT5, RPS10 Methylation, and Ribosome Assembly

APRIL 23, 2010 • VOLUME 285 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 12705

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Jinqi Ren, Yaqing Wang, Yuheng Liang, Yongqing Zhang, Shilai Bao and Zhiheng XuRegulates Ribosome Biogenesis

Methylation of Ribosomal Protein S10 by Protein-arginine Methyltransferase 5

doi: 10.1074/jbc.M110.103911 originally published online February 16, 20102010, 285:12695-12705.J. Biol. Chem. 

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