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PRMT5, Which Forms Distinct Homo-oligomers, Is a Member of the Protein-arginine Methyltransferase Family

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m008660200

ISSN

1083-351X

Autores

Jaerang Rho, Seeyoung Choi, Young Rim Seong, Won‐Kyung Cho, Soo Hyeun Kim, Dong‐Soo Im,

Tópico(s)

Cancer-related gene regulation

Resumo

We found that JBP1, known as a human homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the hepatitis C virus in a yeast two-hybrid screen. Amino acid sequence analysis revealed that Skb1Hs/JBP1 contains conserved motifs ofS-adenosyl-l-methionine-dependent protein-arginine methyltransferases (PRMTs). Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family. Recombinant PRMT5 protein purified from human cells methylated myelin basic protein, histone, and the amino terminus of fibrillarin fused to glutathione S-transferase. Myelin basic protein methylated by PRMT5 contained monomethylated and dimethylated arginine residues. Recombinant glutathione S-transferase-PRMT5 protein expressed in Escherichia coli also contained the catalytic activity. Sedimentation analysis of purified PRMT5 on a sucrose density gradient indicated that PRMT5 formed distinct homo-oligomeric complexes, including a dimer and tetramer, that comigrated with the enzyme activity. The PRMT5 homo-oligomers were dissociated into a monomer in the presence of a reducing agent, whereas a monomer, dimer, and multimer were detected in the absence or at low concentrations of a reducing agent. The results indicate that both covalent linkage by a disulfide bond and noncovalent association are involved in the formation of PRMT5 homo-oligomers. Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract. PRMT5 appears to have lower specific enzyme activity than PRMT1. Although PRMT1 is known to be mainly located in the nucleus, human PRMT5 is predominantly localized in the cytoplasm. We found that JBP1, known as a human homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the hepatitis C virus in a yeast two-hybrid screen. Amino acid sequence analysis revealed that Skb1Hs/JBP1 contains conserved motifs ofS-adenosyl-l-methionine-dependent protein-arginine methyltransferases (PRMTs). Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family. Recombinant PRMT5 protein purified from human cells methylated myelin basic protein, histone, and the amino terminus of fibrillarin fused to glutathione S-transferase. Myelin basic protein methylated by PRMT5 contained monomethylated and dimethylated arginine residues. Recombinant glutathione S-transferase-PRMT5 protein expressed in Escherichia coli also contained the catalytic activity. Sedimentation analysis of purified PRMT5 on a sucrose density gradient indicated that PRMT5 formed distinct homo-oligomeric complexes, including a dimer and tetramer, that comigrated with the enzyme activity. The PRMT5 homo-oligomers were dissociated into a monomer in the presence of a reducing agent, whereas a monomer, dimer, and multimer were detected in the absence or at low concentrations of a reducing agent. The results indicate that both covalent linkage by a disulfide bond and noncovalent association are involved in the formation of PRMT5 homo-oligomers. Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract. PRMT5 appears to have lower specific enzyme activity than PRMT1. Although PRMT1 is known to be mainly located in the nucleus, human PRMT5 is predominantly localized in the cytoplasm. PRMT5, which forms distinct homo-oligomers, is a member of the protein-arginine methyltransferase family.Journal of Biological ChemistryVol. 276Issue 19PreviewIn our paper, we demonstrated that Skb1Hs/JBP1 contains an intrinsic protein-arginine methyltransferase activity. Frankel and Clarke (Frankel, A., and Clarke, S. (2000)J. Biol. Chem. 275, 32794–32982) and we renamed Skb1Hs/JBP1 as PRMT5. Pollack et al. (Pollack, B. P., Kotenko, S. V., He, W., Izotova, L. S., Barnoski, B. L., and Pestka, S. (1999) J. Biol. Chem. 274, 31531–31542) reported for the first time that Skb1Hs/JBP1 contains protein-arginine methyltransferase activity and also suggested that JPB1/PRMT5 forms a homo-oligomer. Full-Text PDF Open Access protein-arginine methyltransferases myelin basic protein hepatitis C virus glutathione S-transferase green fluorescent protein S-adenosyl-l-methionine polyacrylamide gel electrophoresis dithiothreitol high pressure liquid chromatography Protein arginine methylation is an irreversible, post-translational covalent modification. Protein-arginine methyltransferases (PRMTs)1transfer the methyl group fromS-adenosyl-l-methionine to the guanidino nitrogen atoms of an arginine residue (1Gary J.D. Clarke S. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). PRMTs are classified into two major types, I and II, based on substrate and reaction product specificity. Both type I and II PRMTs are common in the formation of monomethylarginine, but the two differ in that type I PRMT catalyzes asymmetric dimethylarginine, whereas type II PRMT produces symmetric dimethylarginine. Type I PRMTs methylate arginines in the Arg-Gly-Gly-rich region, known as the RGG motif, present in many RNA-binding proteins (2Najbauer J. Johnson B.A. Young A.L. Aswad D.W. J. Biol. Chem. 1993; 268: 10501-10509Abstract Full Text PDF PubMed Google Scholar, 3Liu Q. Dreyfuss G. Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (272) Google Scholar, 4Kim S. Merrill B. Rajpurohit R. Kumar A. Stone K. Papov V. Schneiders J. Szer W. Wilson S. Paik W.K. Williams K. Biochemistry. 1997; 36: 5185-5192Crossref PubMed Scopus (108) Google Scholar), or in the Arg-Xaa-Arg motif in poly(A)-binding protein II (5Smith J., J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Lindert M. Herschmann H., R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Myelin basic protein (MBP) and the spliceosomal D1 and D3 proteins are the only known in vivosubstrates for type II PRMT (6Baldwin G.S. Carnegie P.R. Science. 1971; 171: 579-581Crossref PubMed Scopus (148) Google Scholar, 7Stoner G.L. J. Neurochem. 1984; 43: 433-447Crossref PubMed Scopus (98) Google Scholar, 8Brahms H. Raymackers J. Union A. de Keyser F. Meheus L. Luhrmann R. J. Biol. Chem. 2000; 275: 17122-17129Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). However, the classification of type I and II protein-arginine methyltransferases is tenuous because they are based only on substrate and reaction product specificity (1Gary J.D. Clarke S. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). Although five different kinds of genes for protein-arginine methyltransferases PRMT1, HRMT1L1 (human argininemethyltransferase-1L1)/PRMT2, PRMT3, CARM1 (coactivator-associatedargininemethyltransferase-1)/PRMT4, and Skb1Hs (Shk1 kinase-binding protein-1 H omo s apiens)/JBP1 (named PRMT5) have been cloned in mammalian cells, protein-arginine methyltransferase activities of the gene products are demonstrated in only PRMT1, PRMT3, and CARM1/PRMT4 (9Lin W.-J. Gary D.J. Yang M.C. Clarke S. Herschmann H.R. J. Bio. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 10Tang J. Gary D.J. Clarke S. Herschmann H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 11Stallcup M.R. Chen D. Koh S.S. Ma H. Lee Y.-H. Li H. Schurter B.T. Aswad D.W. Biochem. Soc. Trans. 2000; 28: 415-418Crossref PubMed Google Scholar). The gene for rat PRMT1 is the first mammalian gene cloned 30 years after the discovery of protein arginine methylation (12Paik W.K. Kim S. Biochem. Biophys. Res. Commun. 1967; 29: 14-20Crossref PubMed Scopus (121) Google Scholar, 13Paik W.K. Kim S. J. Biol. Chem. 1968; 243: 2108-2114Abstract Full Text PDF PubMed Google Scholar). PRMT1 interacting with the mammalian immediate-early protein (TIS21/PC3), known as BTG2 (14Rouault J.P. Falette N. Guehenneux F. Guillot C. Rimokh R Wang Q. Berthet C. Moyret-Lalle C. Savatier P. Pain B. Shaw P. Berger R. Samarut J. Magaud J.P. Ozturk M. Samarut C. Puisieux A. Nat. Genet. 1996; 14: 482-486Crossref PubMed Scopus (359) Google Scholar, 15Montagnoli A. Guardavaccaro D. Starace G. Tirone F. Cell Growth Differ. 1996; 7: 1327-1336PubMed Google Scholar), is a predominant protein-arginine methyltransferase in mammalian cells and tissues (16Tang J. Frankel A. Cook R.J. Kim S. Paik W.K. Williams K.R. Clarke S. Herschmann H.R. J. Biol. Chem. 2000; 275: 7723-7730Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 17Tang J. Kao P.N. Herschmann H.R. J. Biol. Chem. 2000; 275: 19866-19876Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Subsequently, human PRMT1, which is almost identical to rat PRMT1, was found to be associated with the intracytoplasmic domain of the interferon-α/β receptor (18Abramovich C. Yakobson B. Chebath J. Revel M. EMBO J. 1997; 16: 260-266Crossref PubMed Scopus (154) Google Scholar). The gene for PRMT2 was found by screening expressed sequence tag data bases (19Katsanis N. Yaspo M.-L. Fisher E.M.C. Mamm. Genome. 1997; 8: 526-529Crossref PubMed Scopus (66) Google Scholar, 20Scott H.S. Antonarakis S.E. Lalioti M.D. Rossier C. Silver P.A. Henry M.F. Genomics. 1998; 48: 330-340Crossref PubMed Scopus (144) Google Scholar), but its protein methyltransferase activity has not been detected. PRMT3 containing a zinc finger domain in its amino terminus (21Frankel A. Clarke S. J. Biol. Chem. 2000; 275: 32974-32982Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) was found by conducting a yeast two-hybrid screen using rat PRMT1 as a bait (10Tang J. Gary D.J. Clarke S. Herschmann H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). CARM1/PRMT4 regulates transcription as an interacting molecule with GRIP1 (glucocorticoidreceptor-interactingprotein-1), a p160 family of transcriptional coactivators (22Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2177Crossref PubMed Scopus (1019) Google Scholar). Pollack et al. (23Pollack B.P. Kotenko S.V. He W. Izotova L.S. Barnoski B.L. Pestka S. J. Biol. Chem. 1999; 274: 31531-31542Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) found that Skb1Hs (24Gilbreth M. Yang P. Wang D. Frost J. Polverino A. Cobb M.H. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13802-13807Crossref PubMed Scopus (61) Google Scholar, 25Gilbreth M. Yang P. Bartholomeusz G. Pimental R.A. Kansra S. Gadiraju R. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14781-14786Crossref PubMed Scopus (62) Google Scholar), named JBP1 (Janus kinase-bindingprotein-1), interacts with Janus kinases and contains the protein methyltransferase activity. However, it was not clearly determined whether the protein methylation activity of JBP1 is arginine-specific. Any biological consequences of an interaction between Janus kinases and JBP1 are unknown. Independently, we found that NS3 (nonstructural protein-3) of the hepatitis C virus (HCV) interacts with an Skb1Hs protein, a human homolog of Skb1 (Shk1kinase-bindingprotein-1) of fission yeast, in a yeast two-hybrid screen. HCV causes acute and chronic liver diseases such as liver cirrhosis and hepatocellular carcinoma (26Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6397) Google Scholar, 27Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. Choo Q.L. Houghton M. Kuo G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1086) Google Scholar). The NS3 protein of HCV not only contains serine protease (28Bartenschlager R. Ahlborn-Laake L. Mous J. Jacobson H. J. Virol. 1993; 67: 3835-3844Crossref PubMed Google Scholar, 29Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 2832-2843Crossref PubMed Google Scholar, 30Hijikata M. Mizhushima H. Akagi T. Mori S. Kakiuchi N. Kato N. Tanaka T. Kimura K. Shimotohno K. J. Virol. 1993; 67: 4665-4675Crossref PubMed Google Scholar, 31Tomei L. Failla C. Santolini E. De Franscesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar) and RNA helicase (32Kim D.W. Gwack Y. Han J.H. Choe J. J. Virol. 1997; 71: 9400-9409Crossref PubMed Google Scholar,33Lin C. Kim J.L. J. Virol. 1999; 73: 8798-8807Crossref PubMed Google Scholar) activities, both of which appear to be essential for the virus replication, but also has been implicated in cellular transformation (34Sakamuro D. Furukawa T. Takegami T. J. Virol. 1995; 69: 3893-3896Crossref PubMed Google Scholar, 35Fujita T. Ishido S. Muramatsu S. Itoh M Hotta H. Biochem. Biophys. Res. Commun. 1996; 229: 825-831Crossref PubMed Scopus (82) Google Scholar). For example, NIH3T3 mouse fibroblasts transfected with the N-terminal domain of NS3 become transformed and are tumorigenic in nude mice (34Sakamuro D. Furukawa T. Takegami T. J. Virol. 1995; 69: 3893-3896Crossref PubMed Google Scholar). In fission yeast, Skb1 interacts with the Shk1 kinase, which is a yeast homolog of human p21Cdc42/Rac1-activated kinase (24Gilbreth M. Yang P. Wang D. Frost J. Polverino A. Cobb M.H. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13802-13807Crossref PubMed Scopus (61) Google Scholar). Skb1 has been suggested to regulate mitosis negatively and can be functionally replaced with its human homolog (Skb1Hs) in fission yeast (25Gilbreth M. Yang P. Bartholomeusz G. Pimental R.A. Kansra S. Gadiraju R. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14781-14786Crossref PubMed Scopus (62) Google Scholar). In this context, an interaction between Skb1Hs and NS3 may play a role in liver diseases caused by HCV. To investigate a function of Skb1Hs interacting with the viral protein, its amino acid sequence was compared with those of genes registered in the GenBankTM/EBI Data Bank. The comparison revealed that the C-terminal domain of Skb1Hs contains an extensive homology to a family of proteins with arginine-specific protein methyltransferase activity. In this study, we focused on the biochemical properties of PRMT5 and found that the arginine residue present in MBP is methylated by PRMT5 and that homo-oligomerization is important for the catalytic activity. A homomeric complex of PRMT5 was detected in vivo by co-immunoprecipitation analysis. The homomeric complex of PRMT5 could be separated into a dimer and multimer by sucrose gradient sedimentation. Purified PRMT1 also forms homo-oligomers. PRMT5 forms distinct homo-oligomeric complexes different from those formed by PRMT1, but both covalent and noncovalent associations are involved in the homo-oligomerization of PRMT5 and PRMT1. The homo-oligomeric complexes of PRMT5 and PRMT1, both of which methylate MBP in vitro, may account for the controversial polypeptide compositions of protein-arginine methyltransferases previously purified from cells and tissues (3Liu Q. Dreyfuss G. Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (272) Google Scholar, 36Ghosh S.K. Paik W.K. Kim S. J. Biol. Chem. 1988; 263: 19024-19033Abstract Full Text PDF PubMed Google Scholar, 37Rawal N. Rajpurohit R. Paik W.K. Kim S. Biochem. J. 1994; 300: 483-489Crossref PubMed Scopus (44) Google Scholar). 293T, COS-1, and Chang liver monolayer cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies, Inc.) in an atmosphere of 5% CO2 and air humidified at 37 °C. A yeast two-hybrid screen was conducted using a HeLa cell cDNA library and a bait plasmid that encodes the LexA DNA-binding domain fused to the N-terminal domain (amino acids 1027–1297) of HCV NS3. The isolation of positive clones and subsequent analysis were carried out as previously reported (38Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1333) Google Scholar). One of the positive clones, named clone 4-7, encodes a partial region of human PRMT5 cDNA. To obtain full-length 4-7, primers and a HepG2 cell cDNA library were used in polymerase chain reactions. A partial clone missing ∼200 base pairs from the N terminus of clone 4-7 was obtained and sequenced. The missing N-terminal portion was cloned by reverse transcription-polymerase chain reaction of total mRNAs from HepG2 cells. The full-length PRMT5 cDNA was inserted into the HincII site of pBluescript KS (Stratagene) and sequenced, yielding pBS-4-7F. pFLAG-PRMT5, a FLAG epitope-tagged expression plasmid, was constructed by inserting a 1661-base pairHindIII-XhoI fragment (amino acids 85–637) of PRMT5 cDNA into the HindIII-SalI fragment of a pCMV2-FLAG vector (Sigma) and then inserting the remaining 258-base pair HindIII fragment (amino acids 1–84) of PRMT5 into the 5′-HindIII site. To construct pFLAG-PRMT5-C (where C is the C-terminal region of PRMT5), a 992-base pairPstI-XhoI fragment (amino acids 308–637) of pBS-4-7F was subcloned into pUC19, and then theHindIII-XhoI fragment was subcloned into pCMV2-FLAG. To make a glutathione S-transferase (GST) fusion expression plasmid of PRMT5, a full-length fragment of PRMT5 was inserted into the ClaI-NotI fragment of pEBG (pGST-PRMT5). An amino-terminal region (amino acids 1–309) of PRMT5 was subcloned into the ClaI-NotI fragment of pEBG (pGST-PRMT5-N). A C-terminal region (amino acids 315–637) of PRMT5 was subcloned into the BamHI-NotI fragment of pEBG (pGST-PRMT5-C). To construct the green fluorescent protein (GFP) fusion plasmid pGFP-PRMT5, the BamHI-XhoI fragment of full-length PRMT5 was taken from plasmid pBS-4-7F and subcloned into the BglII-SalI fragment of pEGFP-C1 (CLONTECH). To make a methyltransferase domain I mutant (pGST-PRMT5-M), primers 5′-AAGGATCCACCATGGCGGCGATGGCGGTCGGG-3′, 5′- AAGCGGCGGCCTAGAGGCCAATGGTATATGAGCG-3′, 5′-AACTCGCGTCGCAGCACCATCAGTACCTGGAC-3′, and 5′-AACTCGAGCCCCTGGTGAACGCTTCCCTG-3′ were used in the polymerase chain reactions to amplify a mutant of PRMT5 cDNA. The amplified fragment was subcloned into the BamHI-NotI fragment of pGEX4T-1 (Amersham Pharmacia Biotech). The mutated region was confirmed by sequencing. Full-length PRMT5 and the C-terminal region of PRMT5 (amino acids 315–637) were each cloned into theBamHI-NotI fragment of pGEX4T-1 (yielding pGST-PRMT5 and pGST-PRMT5-C, respectively) for the preparation of GST fusion proteins expressed in Escherichia coli. To construct a FLAG-tagged PRMT1 expression plasmid, pCMV2-PRMT1 (FLAG-PRMT1), rat PRMT1 cDNA was amplified from pGEX(SN)-PRMT1 (9Lin W.-J. Gary D.J. Yang M.C. Clarke S. Herschmann H.R. J. Bio. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) using primers 5′-CCGGATCCACCATGGCGGCAGCCGAGGCCGCG-3′ and 5′-CCGCGGCCGCTCAGCGCATCCGGTAGTCGG-3′. The amplified PRMT1 DNA was subcloned into the BamHI-NotI fragment of pBluescript KS, digested with HindIII and NotI, and inserted into the HindIII-NotI fragment of pCMV2-FLAG. 293T cells (5 × 105 cells/ml) were plated 18–24 h before transfection. Expression plasmids (1–10 μg) were used in the transfections. After 36 h, the cells were lysed with lysis buffer (25 mmTris-HCl (pH 7.5), 150 mm NaCl, 1 mmphenylmethylsulfonyl fluoride, 1% Nonidet P-40, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). The lysates were cleared by centrifugation at 12,000 rpm for 10 min. Anti-FLAG antibody- or glutathione-conjugated agarose beads were added to the cleared lysates and incubated at 4 °C for 2 h with rocking. The beads were washed twice with lysis buffer and twice with PRMT assay buffer (25 mmTris-HCl (pH 7.5), 1 mm EDTA, 1 mm EGTA, and 1 mm phenylmethylsulfonyl fluoride). Proteins bound to the beads or the purified PRMT proteins were then incubated in a 40-μl reaction volume containing 0.25 μCi ofS-[methyl-14C]adenosyl-l-methionine ([14C]AdoMet; specific radioactivity of 56 mCi/mmol; Amersham Pharmacia Biotech) and 0.1–5 μg of methyl acceptors at 30 °C for 2 h. Methylation reactions were stopped by the addition of 2× or 3× SDS-PAGE sample buffer (50 mmTris-HCl (pH 6.8), 100 mm DTT, 2% SDS, 10% glycerol, and 0.1% bromphenol blue). Proteins or methylated proteins were boiled in SDS-PAGE sample buffer at 100 °C for 5 min (except where indicated) and separated on slab gels prepared from 29% (w/v) acrylamide and 1% (w/v)N,N-methylenebisacrylamide (1.5 mm × 5.5 cm-resolving gel) using the buffer system described by Laemmli (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (215638) Google Scholar) at a constant current of 50 mA for ∼2 h. Following electrophoresis, gels were fixed with a fixing solution (45% (v/v) methanol and 10% (v/v) acetic acid) and then soaked in Amplify (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Gels were dried in a vacuum, and radioactivity was visualized by exposing the gels to an x-ray film at −80 °C for 3–14 days or by a Fuji BAS1000 PhosphorImager. 14C incorporation was quantitated using a PhosphorImager. Gels were stained with Coomassie Brilliant Blue R-250 for 20–30 min and destained with the fixing solution to visualize the protein bands. Proteins on the gels were transferred to polyvinylidene difluoride membranes (PerkinElmer Life Sciences). Membranes were blocked with Tris-buffered saline (50 mm Tris-HCl (pH 7.4) and 150 mm NaCl) containing 5% skim milk and then incubated with mouse anti-FLAG or anti-GST antibodies at a concentration of 5 μg/ml. Membranes were washed three times with Tris-buffered saline and incubated with goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma) at 1:5000 dilution. After being washed three times, the reactive proteins were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). The enzyme assay was done in a 500-μl reaction volume containing 80 μg of MBP, 10 μg of purified FLAG-PRMT5, and 1 μCi of [14C]AdoMet. MBP was then precipitated with 500 μl of 25% (w/v) trichloroacetic acid. The precipitate was washed with acetone twice and then dissolved in distilled water. The solution was dried in a 6 × 50-mm glass vial and hydrolyzed with 6 n HCl at 110 °C for 24 h in a Waters Pico-Tag work station (Pico-Tag® System, Waters, Milford, MA). Released amino acids were labeled with phenylisothiocyanate and separated on a Pico-Tag column according to the recommended instructions of the manufacturer. Monomethylated and symmetrically dimethylated arginines purchased from Sigma were also labeled in the same manner and used as standards. The sample and the standards were injected onto an HPLC column (Waters Pico-Tag, 3.9 × 300 mm) equilibrated with 140 mm sodium acetate buffer containing 0.05% (v/v) triethylamine and 6% (v/v) acetonitrile at 46 °C, respectively. Amino acids were eluted from the column with the acetonitrile gradient recommended by the manufacturer. Phenylisothiocyanate- and 14C-labeled amino acids were simultaneously detected on a serially connected UV detector (269 nm) and a Model 150TR on-line flow scintillation analyzer (Packard Instrument Co.), respectively. To detect in vivointeractions between PRMT5 proteins, 293T cells were transfected with various sets of PRMT5 expression plasmids. After transfections, the cells were broken in lysis buffer. Aliquots of cell lysates were incubated with glutathione or anti-FLAG antibody beads for 2 h at 4 °C. The beads were then washed five times with lysis buffer. The bead-bound proteins dissolved in 1× SDS-PAGE sample buffer with a final concentration of 100 mm DTT were then boiled. Protein samples were separated by SDS-PAGE and analyzed by Western blotting with anti-GST or anti-FLAG antibodies. 293T cells (5 × 108) transfected with pCMV2-PRMT5 or pCMV2-PRMT1 DNA were broken in lysis buffer as described above. The cleared lysates were applied to an anti-FLAG affinity column (1 × 10 cm) equilibrated in lysis buffer, and the column was then washed twice with lysis buffer. PRMT proteins were eluted with 100 mm glycine HCl buffer (pH 3.5). The protein elutes were collected in 1 m Tris base buffer (pH 8.0) and then dialyzed against PRMT buffer. The dialysates were concentrated using a Nanospin Plus centrifugal filter (Gelman Instrument Co.). GST fusion proteins were expressed and purified as described by the manufacturer (Amersham Pharmacia Biotech). In brief, cells harboring GST fusion expression plasmids were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 2–3 h at 30 °C. Cells were washed with phosphate-buffered saline buffer, resuspended in lysis buffer, and then sonicated. Soluble protein extracts were loaded onto glutathione-agarose columns (1 × 10 cm). The columns were washed four times with lysis buffer. Bead-bound proteins were eluted with PRMT buffer containing 10 mm reduced glutathione. After dialysis, purified proteins were stored at −70 °C. A GST-PRMT5-C (amino acids 315–637) fusion protein was expressed as a soluble form in E. coli. The fusion protein was purified as described above. The purified fusion protein, which appeared to be homogeneous on an SDS-polyacrylamide gel, was used to immunize BALB/c mice for antibody production. The purified FLAG-PRMT5 or FLAG-PRMT1 protein (20–40 μg) was overlaid on a 35-ml gradient of 5–45% sucrose in sedimentation buffer (50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm EGTA, and 1 mm phenylmethylsulfonyl fluoride). After centrifugation at 10 °C for 24 h at 25,000 rpm in a Beckman SW 28 rotor, the fractions were collected from the bottom. To analyze a homo-oligomer of endogenous PRMT5 in human cells by sedimentation, 293T cells (1 × 108) were washed once with phosphate-buffered saline and resuspended in 3 ml of PRMT buffer. The resuspension was sonicated for 30 s on ice. Cell debris was removed by centrifugation. The soluble protein extract was sedimented on a 35-ml 5–45% sucrose density gradient under the same conditions as described above. Glutaraldehyde (40Matsumoto M. Hwang S.B. Jeng K.-S. Zhu N. Lai M.M.C. Virology. 1996; 218: 43-51Crossref PubMed Scopus (139) Google Scholar) was used to form chemical bridges in the homo-oligomers of FLAG-PRMT5. The purified FLAG-PRMT5 or FLAG-PRMT5-C protein (100 ng) was incubated with glutaraldehyde (0.00006–0.0006%) in 20 μl of PRMT buffer for 5 min at room temperature. The samples were boiled in SDS-PAGE sample buffer containing 100 mm DTT for 5 min and then separated by 6% SDS-PAGE. PRMT5 proteins were detected by Western blotting with an anti-FLAG monoclonal antibody. COS-1 or Chang's liver cells (105 cells/ml) were cultured on a polylysine-coated slide glass before transfection. As a control, pEGFP-PRMT5 or pEGFP-C1 (5 μg) was transfected. After 24 h, the cells were observed with a confocal microscope. Searches of the GenBankTM/EBI Data Bank to identify the biochemical function of PRMT5 revealed that full-size PRMT5 does not have significant homology to any characterized proteins. We separated N- and C-terminal regions of PRMT5 and searched to find whether the N- or C-terminal region has amino acid sequence homology to characterized proteins. The search revealed that whereas the N-terminal region does not have any homology to the known genes, the C-terminal portion of PRMT5 contains amino acid sequences homologous to domains I–III present in AdoMet-utilizing methyltransferases including PRMT1 (9Lin W.-J. Gary D.J. Yang M.C. Clarke S. Herschmann H.R. J. Bio. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar,41Koonin E.V. J. Gen. Virol. 1993; 74: 733-740Crossref PubMed Scopus (204) Google Scholar, 42Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (428) Google Scholar, 43Schluckebier G. O'Gara M. Saenger W. Cheng X. J. Mol. Biol. 1995; 247: 16-20Crossref PubMed Scopus (236) Google Scholar) (Fig. 1). The amino acid sequence of the post-I domain of PRMT5 (Lys-Tyr-Ala-Val-Glu) matches well with that of human PRMT2 (Val-Tyr-Ala-Val-Glu) (19Katsanis N. Yaspo M.-L. Fisher E.M.C. Mamm. Genome. 1997; 8: 526-529Crossref PubMed Scopus (66) Google Scholar, 20Scott H.S. Antonarakis S.E. Lalioti M.D. Rossier C. Silver P.A. Henry M.F. Genomics. 1998; 48: 330-340Crossref PubMed Scopus (144) Google Scholar). Furthermore, intervals between the conserved domains in PRMT5, PRMT1, and PRMT3 are also well preserved. These observations led us to examine the possibility of whether PRMT5 contains intrinsic protein-arginine methyltransferase activity. The GST-PRMT5 plasmid, with GST at the N terminus of the gene for PRMT5, and a control plasmid (GST) were transfected into 293T cells, respectively. The cell lysates were pulled-down with glutathione-agarose beads. The bead suspensions were incubated with [14C]AdoMet and MBP. The reaction products were visualized by SDS-PAGE and fluorography (Fig.2 A). MBP was radioactively labeled only by the beads incubated with the cell lysate transfected with the GST-PRMT5 plasmid, but not by the beads from the control plasmid. The GST-PRMT5 protein bound to the beads was detected by Western blotting with anti-GST antibody (Fig. 2 A). To further demonstrate the protein methyltransferase activity of PRMT5, the FLAG-PRMT5 protein was partially purified from the 293T cell lysate transfected with the FLAG-PRMT5 plasmid via anti-FLAG antibody-conjugated agarose beads. Two closely migrating protein bands of ∼72 kDa in size in the enzyme preparation were detected by Western blotting with anti-PRMT5-C antibody and Coomassie Blue staining (Fig.2 B). The fast migrating band of 72 kDa was not detected by Western blotting with anti-FLAG antibody (data not shown), suggesting that this protein is endogenous PRMT5 and that a homomeric complex between FLAG-PRMT5 and endogenous PRMT5 is formed. MBP was methylated by purified FLAG-PRMT5 in a dose-dependent manner (Fig.2 B, 14 C-M

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