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• W2034229464 abstract "Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3′-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization. Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3′-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization. The 3′-end of nearly all eukaryotic mRNAs harbors a poly(A) tail. Whereas numerous studies indicate that the presence of a poly(A) tail on a eukaryotic mRNA is required for efficient nuclear export, RNA stability, and translational control (1Dreyfus M. Regnier P. Cell. 2002; 111: 611-613Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), recent findings have questioned the direct role of the poly(A) tail in specific steps of mRNA metabolism (2Bird G. Fong N. Gatlin J.C. Farabaugh S. Bentley D.L. Mol. Cell. 2005; 20: 747-758Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 3Dower K. Kuperwasser N. Merrikh H. Rosbash M. RNA. 2004; 10: 1888-1899Crossref PubMed Scopus (96) Google Scholar, 4Meaux S. Van Hoof A. RNA. 2006; 12: 1323-1337Crossref PubMed Scopus (81) Google Scholar). The length of this stretch of polyadenosines is controlled in a species-dependent manner with average lengths of 70 and 250 nucleotides in yeast and mammals, respectively (5Edmonds M. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 285-389Crossref PubMed Google Scholar, 6Mangus D.A. Evans M.C. Jacobson A. Genome Biol. 2003; 4: 223Crossref PubMed Scopus (455) Google Scholar). Yet, the exact mechanism that controls poly(A) tail length remains elusive. mRNA poly(A) tail synthesis involves at least 20 different proteins in the yeast Saccharomyces cerevisiae. The cleavage factors I and II (CF I & II) are first responsible for the cotranscriptional cleavage of the pre-mRNA, which is then followed by the polyadenylation of the upstream fragment by members of the cleavage/polyadenylation factor (CPF) complex, including the poly(A) polymerase Pap1 (7Proudfoot N. O'Sullivan J. Curr. Biol. 2002; 12: R855-857Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 8Wahle E. Ruegsegger U. FEMS Microbiol. Rev. 1999; 23: 277-295Crossref PubMed Google Scholar, 9Zhao J. Hyman L. Moore C. Microbiol. Mol. Biol. Rev. 1999; 63: 405-445Crossref PubMed Google Scholar). In the presence of a protein with poly(A) binding activity, CF I/II, CPF, and Pap1 are sufficient to reconstitute poly(A) tail synthesis de novo on an RNA in vitro (10Chen J. Moore C. Mol. Cell Biol. 1992; 12: 3470-3481Crossref PubMed Scopus (113) Google Scholar, 11Hector R.E. Nykamp K.R. Dheur S. Anderson J.T. Non P.J. Urbinati C.R. Wilson S.M. Minvielle-Sebastia L. Swanson M.S. EMBO J. 2002; 21: 1800-1810Crossref PubMed Scopus (141) Google Scholar, 12Minvielle-Sebastia L. Preker P.J. Wiederkehr T. Strahm Y. Keller W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7897-7902Crossref PubMed Scopus (142) Google Scholar). Despite remarkable similarities in the polyadenylation machinery between yeast and mammals, species-specific factors have been described. Particularly, the product of the pabp2/pabpn1 gene is thought to be specific to metazoans (13Kuhn U. Wahle E. Biochim. Biophys. Acta. 2004; 1678: 67-84Crossref PubMed Scopus (259) Google Scholar), as no apparent yeast homolog has yet been identified. Poly(A)-binding protein (PABP) 3The abbreviations used are: PABP, poly(A)-binding protein; RRM, RNA recognition motif; PRMT, protein arginine methyltransferase; RMT, arginine methyltransferase; OPMD, oculopharyngeal muscular dystrophy; GFP, green fluorescent protein; GST, glutathione S-transferase; PAP, poly(A) polymerase. 2 was originally identified through biochemical enrichment of a polyadenylation stimulatory factor from calf thymus extracts (14Wahle E. Cell. 1991; 66: 759-768Abstract Full Text PDF PubMed Scopus (241) Google Scholar). Mammalian PABP2 is characterized by a putative coiled-coil region, a single RNA recognition motif (RRM), and a C-terminal arginine-rich domain (6Mangus D.A. Evans M.C. Jacobson A. Genome Biol. 2003; 4: 223Crossref PubMed Scopus (455) Google Scholar, 13Kuhn U. Wahle E. Biochim. Biophys. Acta. 2004; 1678: 67-84Crossref PubMed Scopus (259) Google Scholar). The affinity of PAPB2 to synthetic poly(A) tails is in the nanomolar range and requires both the RRM and the C-terminal arginine-rich domain for optimal and specific interaction with poly(A) (15Kuhn U. Nemeth A. Meyer S. Wahle E. J. Biol. Chem. 2003; 278: 16916-16925Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Wahle E. Lustig A. Jeno P. Maurer P. J. Biol. Chem. 1993; 268: 2937-2945Abstract Full Text PDF PubMed Google Scholar). Experiments using in vitro polyadenylation assays led to a model in which PABP2 stimulates processive poly(A) synthesis by direct and simultaneous interactions with the growing poly(A) tail and the poly(A) polymerase (17Kerwitz Y. Kuhn U. Lilie H. Knoth A. Scheuermann T. Friedrich H. Schwarz E. Wahle E. EMBO J. 2003; 22: 3705-3714Crossref PubMed Scopus (124) Google Scholar). Although in vitro experiments have provided information about the biochemical properties of PABP2, little is known about the mechanism by which it regulates poly(A) tail synthesis in vivo. A Drosophila system was recently established to address the function of PABP2 in vivo. These studies provided evidence for the critical role of PABP2 during embryonic development as well as an unsuspected role in the regulation of cytoplasmic polyadenylation (18Benoit B. Mitou G. Chartier A. Temme C. Zaessinger S. Wahle E. Busseau I. Simonelig M. Dev. Cell. 2005; 9: 511-522Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). An understanding of the in vivo mechanism by which PABP2 regulates polyadenylation is significant, because the human genetic disorder oculopharyngeal muscular dystrophy (OPMD) is linked to mutations in the pabp2 gene (19Brais B. Bouchard J.P. Xie Y.G. Rochefort D.L. Chretien N. Tome F.M. Lafreniere R.G. Rommens J.M. Uyama E. Nohira O. Blumen S. Korczyn A.D. Heutink P. Mathieu J. Duranceau A. Codere F. Fardeau M. Rouleau G.A. Nat. Genet. 1998; 18: 164-167Crossref PubMed Scopus (644) Google Scholar). A physiological hallmark of this disorder is the accumulation of fibrous inclusions in the nuclei of skeletal muscle fibers that consist of PABP2 aggregates (20Calado A. Tome F.M. Brais B. Rouleau G.A. Kuhn U. Wahle E. Carmo-Fonseca M. Hum. Mol. Genet. 2000; 9: 2321-2328Crossref PubMed Scopus (203) Google Scholar). Yet, the molecular defects in PABP2 function that lead to the establishment of this disease remain unclear. Mammalian PABP2 is also subject to a post-translational modification where specific arginine residues are methylated (21Smith J.J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). How arginine methylation modulates the function of PABP2 as well as the cellular methyltransferase responsible for PABP2 methylation remains unknown. Protein arginine methyltransferases (PRMT) catalyze the monomethylation of specific arginine residues in proteins using S-adenosyl-l-methionine as a methyl donor. PRMTs are divided into two major classes depending on the type of dimethylarginine they generate: type I PRMTs modify proteins by the catalysis of asymmetric NG-NG-dimethylarginine, whereas type II PRMTs catalyze the formation of symmetric NG-NG-dimethylarginine (22Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar). prmt-encoding genes have been identified from the sequenced genomes of yeast, worms, flies, plants, and mammals; but not prokaryotes. The biological role of PRMTs is likely mediated by the modification of substrate proteins. Accordingly, extensive large scale efforts have aimed to identify arginine methylated proteins (23Ong S.E. Mittler G. Mann M. Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (368) Google Scholar, 24Lee J. Bedford M.T. EMBO Rep. 2002; 3: 268-273Crossref PubMed Scopus (187) Google Scholar, 25Boisvert F.M. Cote J. Boulanger M.C. Richard S. Mol. Cell Proteomics. 2003; 2: 1319-1330Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Based on these and other studies, arginine methylation appears to impact a variety of cellular processes including ribosome biosynthesis (26Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar), T-cell activation (27Blanchet F. Cardona A. Letimier F.A. Hershfield M.S. Acuto O. J. Exp. Med. 2005; 202: 371-377Crossref PubMed Scopus (81) Google Scholar), cytokine and interferon signaling (28Mowen K.A. Schurter B.T. Fathman J.W. David M. Glimcher L.H. Mol. Cell. 2004; 15: 559-571Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), cell differentiation (29Balint B.L. Szanto A. Madi A. Bauer U.M. Gabor P. Benko S. Puskas L.G. Davies P.J. Nagy L. Mol. Cell Biol. 2005; 25: 5648-5663Crossref PubMed Scopus (51) Google Scholar), and DNA repair (30Boisvert F.M. Dery U. Masson J.Y. Richard S. Genes Dev. 2005; 19: 671-676Crossref PubMed Scopus (168) Google Scholar). The role of arginine methylation in these cellular pathways is likely regulated by biochemical activities such as protein-protein interactions (31Friesen W.J. Massenet S. Paushkin S. Wyce A. Dreyfuss G. Mol. Cell. 2001; 7: 1111-1117Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 32Cote J. Richard S. J. Biol. Chem. 2005; 280: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), protein subcellular localization (33Cote J. Boisvert F.M. Boulanger M.C. Bedford M.T. Richard S. Mol. Biol. Cell. 2003; 14: 274-287Crossref PubMed Scopus (214) Google Scholar, 34Green D.M. Marfatia K.A. Crafton E.B. Zhang X. Cheng X. Corbett A.H. J. Biol. Chem. 2002; 277: 7752-7760Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), transcription and chromatin remodeling (35Huang S. Litt M. Felsenfeld G. Genes Dev. 2005; 19: 1885-1893Crossref PubMed Scopus (180) Google Scholar, 36Kwak Y.T. Guo J. Prajapati S. Park K.J. Surabhi R.M. Miller B. Gehrig P. Gaynor R.B. Mol. Cell. 2003; 11: 1055-1066Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), mRNA metabolism (37Boisvert F.M. Cote J. Boulanger M.C. Cleroux P. Bachand F. Autexier C. Richard S. J. Cell Biol. 2002; 159: 957-969Crossref PubMed Scopus (151) Google Scholar, 38Yu M.C. Bachand F. McBride A.E. Komili S. Casolari J.M. Silver P.A. Genes Dev. 2004; 18: 2024-2035Crossref PubMed Scopus (110) Google Scholar), and translation (26Bachand F. Silver P.A. EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar, 39Swiercz R. Person M.D. Bedford M.T. Biochem. J. 2005; 386: 85-91Crossref PubMed Scopus (131) Google Scholar). Not unexpectedly, mice engineered for deletion of the prmt1 (40Pawlak M.R. Scherer C.A. Chen J. Roshon M.J. Ruley H.E. Mol. Cell Biol. 2000; 20: 4859-4869Crossref PubMed Scopus (280) Google Scholar), prmt4 (41Yadav N. Lee J. Kim J. Shen J. Hu M.C. Aldaz C.M. Bedford M.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6464-6468Crossref PubMed Scopus (240) Google Scholar), or prmt5 (42Boisvert F.M. Chenard C.A. Richard S. Sci. STKE. 2005; 2005: re2PubMed Google Scholar) gene are inviable. Yet, our understanding of the biological role of each PRMT and how arginine methylation alters the biological function of proteins is limited by the few physiological substrates identified to date. In this study, we report the identification of a nuclear poly(A)-binding protein (Pab2) in fission yeast. Our results reveal that pab2-null cells produce hyperadenylated mRNAs and are cold-sensitive. We also demonstrate that the protein arginine methyltransferase 1 (RMT1) is the enzyme responsible for Pab2 arginine methylation. Experiments to determine the effect of methylation on Pab2 function indicate that the oligomerization levels of Pab2 are increased when it is not methylated. Accordingly, we found that the growth inhibitory effect caused by Pab2 overexpression is exacerbated in rmt1Δ cells. Our results thus establish Pab2 as an important regulator of poly(A) tail length control and support a model in which the oligomerization-dependent cellular toxicity of Pab2 is modulated by arginine methylation. Strains, Growth Media, and Genetic Methods—The complete list of the Schizosaccharomyces pombe strains used in this study is given in Table 1. Cells were grown at 30 °C in yeast extract medium with amino acid supplements (YES) and Edinburgh minimum medium (EMM) containing appropriate amino acid supplements. S. pombe cells were transformed with plasmids and PCR products by the lithium acetate method. Disruption of pab2 was performed by PCR-mediated gene targeting using 100-nt oligonucleotides with 80-nt from the appropriate regions of the pab2 genomic sequence (43Bahler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie 3rd, A. Steever A.B. Wach A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 943-951Crossref PubMed Scopus (1771) Google Scholar). The oligonucleotide sequences used for the construction of these strains are available upon request. Following transformation of a diploid strain, G418-resistant colonies were screened for pab2 heterozygosity by colony-PCR. Meiosis and sporulation were induced in selected heterozygote diploids by plating on malt extract agar and tetrads were dissected with a micromanipulator (MSM 200, Singer Instruments). nmt1+-dependent gene expression was repressed by the addition of 15 μm thiamine to the growth medium.TABLE 1S. pombe strains used in this studyStrainGenotypeRef.FBY13h+ ade6M210 leu1-32 ura4Δ18 his3Δ126FBY1h+ ade6M210 leu1-32 ura4Δ18 his3Δ1 RMT1Δ::KanMX626FBY2h+ ade6M210 leu1-32 ura4Δ18 his3Δ1 RMT3Δ::KanMX626FBY3h+ ade6M210 leu1-32 ura4Δ18 his3Δ1 RMT5Δ::KanMX626FBY106h+ ade6 leu1-32 ura4Δ18 his3Δ1 (spore A)This studyFBY107h− ade6 leu1-32 ura4Δ18 his3Δ1 pab2::kanMX6 (spore B)This studyFBY108h− ade6 leu1-32 ura4Δ18 his3Δ1 (spore C)This studyFBY109h+ ade6 leu1-32 ura4Δ18 his3Δ1 pab2::kanMX6 (spore D)This study Open table in a new tab Plasmid Constructs—The cDNAs encoding S. pombe pab2 and pab2ΔC28 (amino acid 1-138) were amplified by RT-PCR using total cellular RNA extracted from fission yeast. The amplifications were performed using the same forward primer 5′-CCTAGCTAGCAGTGATCAAGATGCCTTAGA-3′ and the 5′-CGCGGATCCCTAATACGGAGCGAAACCACG-3′ and 5′-CGCGGATCCCTAGCTCATACCAGGAACGTTTGTCC-3′ reverse primers, respectively. Following digestion of the 5′- and 3′-ends of the cDNAs with NheI and BamHI, respectively, the cDNAs were cloned into NheI/BamHI-digested pREP2 and pREP82 vectors (44Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (931) Google Scholar) that contain the DNA sequence encoding for a single Myc epitope tag. The pab2 cDNA was also cloned into BamHI-digested pREP41EGFP-N (45Craven R.A. Griffiths D.J. Sheldrick K.S. Randall R.E. Hagan I.M. Carr A.M. Gene (Amst.). 1998; 221: 59-68Crossref PubMed Scopus (200) Google Scholar) using the primer pair 5′-CGCGGATCCGAGTGATCAAGATGCCTTAGA-3′ and 5′-CGCGGATCCCTAATACGGAGCGAAACCACG-3′. pGEX4T2-Pab2 and pGEX4T2-Pab2ΔC28 were generated by PCR-amplification using pREP82myc-PAB2 as a template and the forward primer 5′-CGCGGATCCAGTGATCAAGATGCCTTAGA-3′ and the reverse primers 5′-CGCGGATCCCTAATACGGAGCGAAACCACG-3′ and 5′-CGCGGATCCCTAGCTCATACCAGGAACGTTTGTCC-3′, respectively. pREP41EGFPN-RMT5 was generated by RT-PCR amplification of the rmt5 cDNA using the primer pair 5′-CGCGGATCCGTTATTGCGGGATGGCCGT-3′ and 5′-CGCGGATCCTTAATACATATTACACGAG-3′ both containing BamHI restriction sites. The PCR-amplified sequences were verified by automated sequencing at the University of Calgary DNA Core Facility. Antibodies—Rabbit polyclonal antibodies specific to fission yeast RMT1 and RMT3 were raised at Covance Research Products (Denver, PA) against GST fusion proteins purified from Escherichia coli. Rabbit polyclonal Myc antibody A-14 and the mouse monoclonal anti-GST B-14 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Asymmetric dimethylarginine-specific rabbit polyclonal antibody (ASYM24) was from Upstate (Charlottesville, VA) and the rabbit polyclonal anti-GFP was from Invitrogen (Burlington, ON, Canada). Immunoprecipitation Experiments—30-50 ml of mid-log phase fission yeast cells grown in either YES or EMM were used for immunoprecipitation experiments. Cells were lysed in ice-cold PBS-MT (1× phosphate-buffered saline supplemented with 2 mm MgCl2 and 1% Triton-X-100) containing a mixture of protease inhibitors (Roche Applied Science) with a Fastprep FP120 (Thermo Electro Corp.) using 0.5-mm glass beads. Clarified lysates were normalized for total protein concentration using the Bradford protein assay (Bio-Rad), and 1 mg of total proteins was subjected to immunoprecipitation using agaroseconjugated anti-Myc (9E10; Santa Cruz Biotechnology). Immunoprecipitated proteins were eluted in sample buffer, separated on 12% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting. Microscopy—For localization of GFP-Pab2, FBY13, and FBY1, cells that were previously transformed with pREPEGFPN-Pab2 were grown to saturation in EMM containing 15 μm thiamine. Cells were then washed twice to remove the thiamine and allowed to grow for 18-20 h before direct fluorescence microscopy. The nuclei of cells were stained using Hoechst 33342 (Sigma). Recombinant Protein Expression and in Vitro Pull-down Assays—GST, GST-Pab2, and GST-Pab2ΔC28 were expressed in E. coli BL21 DE3 cells (Invitrogen). Protein expression was induced by the addition of 0.5 mm isopropyl-1-β-d-thiogalactopyranoside for 3 h at 37 °C for GST and GST-Pab2ΔC28, and for 18 h at 18 °C for GST-Pab2. Following centrifugation of the cells and subsequent washing in ice-cold phosphate-buffered saline, the bacterial cells were broken by sonication, and the cell membranes were solubilized by the addition of Triton X-100 to a final concentration of 1%. The cell debris was sedimented by a 10-min centrifugation at 12,000 rpm at 4 °C, and the clarified lysates were subjected to glutathione-Sepharose resin (Amersham Biosciences). After extensive washing, the proteins were eluted from the resin by incubating with 10 mm reduced glutathione resuspended in Tris-HCl pH 8.6; 250 mm NaCl; 0.1% Triton X-100. The proteins were aliquoted and stored at -80 °C. For poly(A) pull-down experiments, 2 mg of recombinant protein or 0.5 mg of total cell extracts were incubated for 1 h at 4 °C with 15 μl of poly(A)-Sepharose 4B (Sigma) that had been previously pre-equilibrated in buffer A (50 mm Tris-HCl, pH 8.0; 150 mm NaCl; 0.1% Triton-X-100; 2 mm MgCl2;1mm dithiothreitol; 1 mm EDTA). The beads were then washed three times with 1 ml of buffer A, and the bound proteins eluted by incubating for 5 min at 95 °C in SDS-PAGE sample buffer. For the oligonucleotide polymers competition experiment, 10-fold excess of polyadenylic or polycytidylic acids (Sigma) were added to poly(A)-Sepharose simultaneously with GST-Pab2. The copurification of the different proteins with poly(A)-Sepharose was analyzed by immunoblotting. RNA Analyses—cDNA synthesis from fission yeast total RNA was as previously described (46Bachand F. Lackner D.H. Bahler J. Silver P.A. Mol. Cell Biol. 2006; 26: 1731-1742Crossref PubMed Scopus (39) Google Scholar) with the exception that the Omniscript reverse transcriptase (Qiagen) was used. cDNAs were PCR-amplified with TaqDNA polymerase (NEB) using the following oligonucleotides sets: forward 5′-CCTAGCTAGCAGTGATCAAGATGCCTTAGA-3′ and reverse 5′-CGCGGATCCCTAATACGGAGCGAAACCACG-3′ primers for pab2, and forward 5′-CCATGTTTTGCGCTAGAGCAGGC-3′ and reverse 5′-CTTCTGAAACAGGCTCGCGAT-3′ primers for rmt1. Poly(A) tail length analyses were based on a previously described method (47Minvielle-Sebastia L. Winsor B. Bonneaud N. Lacroute F. Mol. Cell Biol. 1991; 11: 3075-3087Crossref PubMed Scopus (132) Google Scholar). Briefly, 1 μg of total fission yeast RNA was 3′-end-labeled at 4 °C for 18-20 h with 25 μCi of [32P]cytidine biphosphate using T4 RNA ligase (Ambion). Following digestion of RNAs with RNases T1 and A, the remaining poly(A) tails were ethanol precipitated after proteinase K and phenol-choloform treatments. Poly(A) tracts were separated on 8% acrylamide-7 m urea gels and analyzed using a STORM 860 instrument (Molecular Dynamics). A Gene Product Similar to Nuclear Poly(A)-binding Proteins Copurifies with the Fission Yeast Protein Arginine Methyltransferase 1 (RMT1)—An affinity purification approach was used to identify novel RMT1-binding proteins in the fission yeast S. pombe. Homologous recombination-mediated gene tagging generated a strain that expresses C-terminal TAP-tagged RMT1 from its endogenous promoter. Following tandem purification and analysis of the eluted protein mixture by mass spectrometry, peptides corresponding to several RNA recognition motif (RRM)-containing proteins were identified (Table 2). Inspection of the amino acid sequence of the uncharacterized spbc16e6.12c gene revealed extensive similarity to nuclear poly(A)-binding proteins: a predicted coiled-coil domain consisting of regularly spaced aliphatic residues, a single RNP-type RRM, and a C-terminal arginine-rich domain (Fig. 1). Similar to the Drosophila nuclear poly(A)-binding protein (PABP2), the S. pombe spbc16e6.12c gene product is considerably shorter than metazoan PABP2 proteins because of the lack of an N-terminal region rich in alanine, glycine, glutamic acid, and proline residues. Fission yeast SPBC16E6.12c shares 47% identity and 66% similarity with human PABP2. We therefore named the S. pombe spbc16e6.12c gene pab2 on the basis of this homology. Sequence searches did not find any pab2 ortholog in the S. cerevisiae genome.TABLE 2Proteins identified by LC-MS/MS from RMT1-TAP purificationS. pombeORF numberMWnaNumber of unique tryptic peptides identified from each protein by mass spectrometry.kDaRMT1SPAC890.07c3946SPBC16E9.12cSPBC16E9.12c208Tif31/p135SPBC530.06c1326rpL7SPBC18H10.12c295Gar1SPBC25H2.01c204Bcr1SPBC582.05c984Rad18SPCC5E4.061304rpL32SPBC16C6.11153Gar2SPAC140.02532a Number of unique tryptic peptides identified from each protein by mass spectrometry. Open table in a new tab A construct expressing Myc-tagged Pab2 was generated to validate our large-scale affinity purification results and examine whether RMT1 could coimmunoprecipitate with S. pombe Pab2. The Myc-Pab2 construct was used to transform wild-type as well as cells in which the genomic copy of the rmt1, rmt3, or rmt5 arginine methyltransferase-encoding gene was deleted. Total cells extracts prepared from the different yeast cultures were subjected to immunoprecipitation using a monoclonal Myc antibody and analyzed by immunoblotting using various antibodies. As can be seen in Fig. 2, RMT1, but not RMT3, was found to coprecipitate with Myc-Pab2 in extracts prepared from wild-type cells (lane 7). As controls, Myc immunoprecipitates prepared from extracts of vector-transformed (lane 6) and rmt1-null cells that expressed Myc-tagged Pab2 (lane 8) did not recover RMT1. Deletion of the rmt3 or rmt5 coding sequence did not perturb the association between RMT1 and Pab2 (Fig. 2; lanes 9 and 10, respectively). These results are consistent with our tandem affinity purification data and indicate that the arginine methyltransferase RMT1 associates with Pab2 in fission yeast. Pab2 Is Asymmetrically Dimethylated by RMT1—The C-terminal arginine-rich region of human PABP2 is dimethylated at arginines by PRMT1 and PRMT3 in vitro (21Smith J.J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar); yet, the physiological methyltransferase responsible for PABP2 methylation remains to be determined. Given the specific association between RMT1 and Pab2 (Fig. 2), we examined whether Pab2 was arginine methylated in fission yeast. Extracts of cells that were previously transformed with an empty vector or a vector expressing Myc-Pab2 were subjected to immunopurification using a Myc monoclonal antibody. Fig. 3A demonstrates that similar levels of Myc-Pab2 were immunoprecipitated from wild-type (lane 2; upper panel) and arginine methyltransferases-null cell extracts (lanes 3-5; upper panel), but not from vector control-transformed cells (lane 1; upper panel). An affinity-purified antibody specific for asymmetric dimethylarginines (aDMA) (33Cote J. Boisvert F.M. Boulanger M.C. Bedford M.T. Richard S. Mol. Biol. Cell. 2003; 14: 274-287Crossref PubMed Scopus (214) Google Scholar) was used to determine the methylation status of immunopurified Pab2. Reprobing of the membrane used for the Myc immunoblot with the aDMA-specific antibody detected aDMA-modified Pab2 in immunoprecipitates prepared from extracts of wild-type, rmt3Δ, and rmt5Δ cells (lanes 2, 4, and 5, respectively; lower panel), but not from rmt1-null cells (lane 3; lower panel). Immunoblotting with a symmetric dimethylarginine-specific antibody (37Boisvert F.M. Cote J. Boulanger M.C. Cleroux P. Bachand F. Autexier C. Richard S. J. Cell Biol. 2002; 159: 957-969Crossref PubMed Scopus (151) Google Scholar) did not detect such modification in Myc-Pab2 (data not shown). These results establish RMT1 as the physiological methyltransferase responsible for Pab2 arginine methylation. Furthermore, our data reveal that no alternate pathways exist to complement Pab2 methylation in the absence of RMT1 in S. pombe. We next set out to identify the region in Pab2 that is arginine methylated. The arginine-rich domain has been shown to be the methylation site of several nucleic acid-binding proteins (48Gary J.D. Clarke S. Prog. Nucleic Acids Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar), including human PABP2 (21Smith J.J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We therefore generated a construct that expresses a C-terminal truncated version of Myc-Pab2 lacking the arginine-rich domain. As can be seen in Fig. 3B, the C-terminal-truncated form of Myc-Pab2 expressed in wild-type cells was not found to contain methylated arginine (lane 3; lower panel); yet, both full-length and truncated versions of Myc-Pab2 were successfully immunopurified (lanes 2-3, upper panel). These results define the C-terminal arginine-rich domain as the major site of arginine methylation in fission yeast Pab2. Fission Yeast Pab2 Is a Nonessential Nuclear Protein—We constructed a diploid strain in which one of the two pab2 alleles was disrupted to address whether pab2 is an essential gene in fission yeast. Germination of the spores after meiosis and tetrad microdissection resulted in a 2:2 segregation ratio of geneticin resistance (Fig. 4A), indicating that pab2-null cells are viable in S. pombe. RT-PCR was used to confirm the absence of pab2 expression in geneticin-resistant cells. Analysis of total RNA prepared from cells derived from the dissection of tetrad 6 (Fig. 4A) demonstrated that geneticin-resistant cells from" @default.
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• W2034229464 title "Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast" @default.
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