Alternative Splicing Yields Protein Arginine Methyltransferase 1 Isoforms with Distinct Activity, Substrate Specificity, and Subcellular Localization

PRMT1 is the predominant member of a family of protein arginine methyltransferases (PRMTs) that have been implicated in various cellular processes, including transcription, RNA processing, and signal transduction. It was previously reported that the human PRMT1 pre-mRNA was alternatively spliced to yield three isoforms with distinct N-terminal sequences. Close inspection of the genomic organization in the 5′-end of the PRMT1 gene revealed that it can produce up to seven protein isoforms, all varying in their N-terminal domain. A detailed biochemical characterization of these variants revealed that unique N-terminal sequences can influence catalytic activity as well as substrate specificity. In addition, our results uncovered the presence of a functional nuclear export sequence in PRMT1v2. Finally, we find that the relative balance of PRMT1 isoforms is altered in breast cancer. PRMT1 is the predominant member of a family of protein arginine methyltransferases (PRMTs) that have been implicated in various cellular processes, including transcription, RNA processing, and signal transduction. It was previously reported that the human PRMT1 pre-mRNA was alternatively spliced to yield three isoforms with distinct N-terminal sequences. Close inspection of the genomic organization in the 5′-end of the PRMT1 gene revealed that it can produce up to seven protein isoforms, all varying in their N-terminal domain. A detailed biochemical characterization of these variants revealed that unique N-terminal sequences can influence catalytic activity as well as substrate specificity. In addition, our results uncovered the presence of a functional nuclear export sequence in PRMT1v2. Finally, we find that the relative balance of PRMT1 isoforms is altered in breast cancer. Protein structural and functional diversity is reliant on the covalent posttranslational modification of its amino acid residues. One of these modifications, termed arginine methylation, results from the transfer of methyl groups from S-adenosyl-l-methionine to the guanidino nitrogen atom of arginine residues in protein substrates (1Gary J.D. Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). The protein arginine methyltransferases (PRMTs) 3The abbreviations used are: PRMT, protein arginine methyltransferase; hnRNP, heterogeneous nuclear ribonucleoprotein; NES, nuclear export sequence; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; RT, reverse transcription; siRNA, small interfering RNA; PBS, phosphate-buffered saline; EST, Expressed Sequence Tag; ss, splice site; nt, nucleotides; TSS, transcriptional start site; AdoMet, S-adenosyl-l-methionine; ES, embryonic stem. 3The abbreviations used are: PRMT, protein arginine methyltransferase; hnRNP, heterogeneous nuclear ribonucleoprotein; NES, nuclear export sequence; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; RT, reverse transcription; siRNA, small interfering RNA; PBS, phosphate-buffered saline; EST, Expressed Sequence Tag; ss, splice site; nt, nucleotides; TSS, transcriptional start site; AdoMet, S-adenosyl-l-methionine; ES, embryonic stem. responsible for this process consist of a family of nine enzymes that have been classified as either type I (PRMT1-4, -6, and -8) or type II (PRMT5, -7, and -9) according to whether they promote the formation of asymmetric ω-NG,NG-dimethylarginines or symmetric ω-NG,N′G-dimethylarginines, respectively. An increasing number of PRMT targets have recently been identified that are involved in a broad range of cellular processes, including DNA repair, transcriptional regulation, RNA processing, and signal transduction (2Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). PRMT1 is the most abundant PRMT and accounts for >85% of the asymmetrically dimethylated arginines generated in mammalian cells (3Tang J. Frankel A. Cook R.J. Kim S. Paik W.K. Williams K.R. Clarke S. Herschman H.R. J. Biol. Chem. 2000; 275: 7723-7730Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). PRMT1 activity has mainly been linked with the regulation of protein-protein interactions in signal transduction pathways and cell growth. For example, arginine methylation of Sam68 is required for its proper localization (4Cote J. Boisvert F.M. Boulanger M.C. Bedford M.T. Richard S. Mol. Biol. Cell. 2003; 14: 274-287Crossref PubMed Scopus (212) Google Scholar) and differentially regulates its interaction with SH3 and WW domains (5Bedford M.T. Frankel A. Yaffe M.B. Clarke S. Leder P. Richard S. J. Biol. Chem. 2000; 275: 16030-16036Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Recent observations have shown that arginine methylation of the proline-rich domains of the heterogeneous nuclear ribonucleoprotein K (hnRNP K) also regulates its interaction with the SH3 domain of the tyrosine kinase c-Src (6Ostareck-Lederer A. Ostareck D.H. Rucknagel K.P. Schierhorn A. Moritz B. Huttelmaier S. Flach N. Handoko L. Wahle E. J. Biol. Chem. 2006; 281: 11115-11125Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The hnRNPs represent a major protein family that contains ω-NG,NG-dimethylarginines in vivo (7Liu Q. Dreyfuss G. Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (268) Google Scholar), and arginine methylation has also been shown to regulate the nucleocytoplasmic localization of some of its members (8Nichols R.C. Wang X.W. Tang J. Hamilton B.J. High F.A. Herschman H.R. Rigby W.F. Exp. Cell Res. 2000; 256: 522-532Crossref PubMed Scopus (145) Google Scholar). Surprisingly, very little is known about how arginine methylation is regulated. PRMT1 was first isolated through its interaction with BTG1 and BTG2, two members of the BTG/Tob family of proteins involved in negative regulation of cell growth (9Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 10Rouault 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 (348) Google Scholar, 11Berthet C. Guehenneux F. Revol V. Samarut C. Lukaszewicz A. Dehay C. Dumontet C. Magaud J.P. Rouault J.P. Genes Cells. 2002; 7: 29-39Crossref PubMed Scopus (73) Google Scholar, 12Duriez C. Moyret-Lalle C. Falette N. El-Ghissassi F. Puisieux A. Bull. Cancer. 2004; 91: 242-253PubMed Google Scholar, 13Tirone F. J. Cell. Physiol. 2001; 187: 155-165Crossref PubMed Scopus (194) Google Scholar, 14Boiko A.D. Porteous S. Razorenova O.V. Krivokrysenko V.I. Williams B.R. Gudkov A.V. Genes Dev. 2006; 20: 236-252Crossref PubMed Scopus (115) Google Scholar, 15Lim I.K. J. Cancer Res. Clin. Oncol. 2006; 132: 417-426Crossref PubMed Scopus (88) Google Scholar). BTG2 is a direct substrate of PRMT1 (16Lim I.K. Park T.J. Kim S. Lee H.W. Paik W.K. Biochem. Mol. Biol. Int. 1998; 45: 871-878PubMed Google Scholar), and both BTG1 and -2 can stimulate its activity (9Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 17Bakker W.J. Blazquez-Domingo M. Kolbus A. Besooyen J. Steinlein P. Beug H. Coffer P.J. Lowenberg B. von Lindern M. van Dijk T.B. J. Cell Biol. 2004; 164: 175-184Crossref PubMed Scopus (140) Google Scholar). Recent observations have also suggested that PRMT1 activity could be regulated by its dynamic subcellular localization (18Herrmann F. Lee J. Bedford M.T. Fackelmayer F.O. J. Biol. Chem. 2005; 280: 38005-38010Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). As an example, the protein hCAF1 (CCR4-associated factor 1), a known interactor of BTG1, has been shown to form a complex with PRMT1 in nuclear speckles and to regulate its activity in a substrate-dependent manner (19Robin-Lespinasse Y. Sentis S. Kolytcheff C. Rostan M.C. Corbo L. Le Romancer M. J. Cell Sci. 2007; 120: 638-647Crossref PubMed Scopus (57) Google Scholar). Although all PRMTs contain a highly conserved methyltransferase domain of approximately 310 amino acids, each PRMT has distinct protein substrate specificities. Beyond their “core” region, each PRMT exhibits a unique N-terminal region that varies considerably in length. The function of this PRMT-specific sequence is still not well understood, but results from deletion studies suggest that it can contribute to PRMT enzymatic activity and/or protein substrate recognition. For example, deletion of the N terminus of PRMT3, which contains a zinc-finger motif, decreases its enzymatic activity and affects its protein substrate specificity (20Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 21Frankel A. Clarke S. J. Biol. Chem. 2000; 275: 32974-32982Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Similarly, deletion of the N-terminal part of Hmt1, the yeast PRMT1 homologue, significantly impairs its oligomerization and reduces its methyltransferase activity both in vivo and in vitro (22Weiss V.H. McBride A.E. Soriano M.A. Filman D.J. Silver P.A. Hogle J.M. Nat. Struct. Biol. 2000; 7: 1165-1171Crossref PubMed Scopus (528) Google Scholar). It was previously reported that alternative splicing of the human PRMT1 primary transcript could generate at least three protein isoforms, themselves differing at their N terminus (23Scott 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, 24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar). We describe here the complex genomic organization in the 5′-end of the PRMT1 gene that can produce up to seven protein isoforms expressed in a tissue-specific manner. Biochemical characterization of these seven isoforms (designated as v1 to v7) revealed that they are all active, except for v7, and that their unique N-terminal sequences can confer distinct substrate specificity. Moreover, we demonstrate that the amino acid sequence unique to v2 (encoded by exon 2) contains a CRM1-dependent nuclear export sequence (NES) that regulates its subcellular localization. Finally, we find that the relative balance of these PRMT1 splicing variants is altered in breast cancer cell lines. Cells, Reagents, and Antibodies—The human HeLa cervical carcinoma cell line was purchased from ATCC (Manassas, Virginia) and grown as a monolayer in Dulbecco's modified Eagle's medium supplemented with 1 mm sodium pyruvate, 50 IU/ml penicillin, 50 mg/ml streptomycin, and 10% fetal calf serum (Wisent). The human normal breast cell line Hs 578 Bst and the human breast cancer cell lines Hs 578T, BT-20, BT-549, MCF7, MDA-MB-231, and T-47D were purchased from ATCC and grown as monolayers in complete Dulbecco's modified Eagle's medium supplemented with minimum Eagle's medium nonessential amino acids and 10% fetal bovine serum (Wisent). Mouse wild type and PRMT1-/- ES cells were kindly provided by Dr. David Lohnes (University of Ottawa, ON, Canada) and Dr. H. Earl Ruley (Vanderbilt University Medical Center, Nashville, TN), respectively, and were maintained as previously described (25Pawlak M.R. Scherer C.A. Chen J. Roshon M.J. Ruley H.E. Mol. Cell. Biol. 2000; 20: 4859-4869Crossref PubMed Scopus (278) Google Scholar). Transfections were performed using the Lipofectamine Plus transfection reagent (Invitrogen) according to manufacturer's instructions. Polyclonal anti-GFP antibodies were purchased from Sigma. Rabbit polyclonal antibodies against human PRMT1, Sam68, and asymmetric dimethylarginine (ASYM24-25) were described previously (4Cote J. Boisvert F.M. Boulanger M.C. Bedford M.T. Richard S. Mol. Biol. Cell. 2003; 14: 274-287Crossref PubMed Scopus (212) Google Scholar, 26Chen T. Boisvert F.M. Bazett-Jones D.P. Richard S. Mol. Biol. Cell. 1999; 10: 3015-3033Crossref PubMed Scopus (120) Google Scholar, 27Boisvert F.M. Cote J. Boulanger M.C. Richard S. Mol. Cell. Proteomics. 2003; 2: 1319-1330Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) and were a kind gift from Dr. Stephane Richard (McGill University, Montreal, Qc, Canada). Monoclonal antibodies against β-actin and glyceraldehyde-3-phosphate dehydrogenase were purchased from Sigma and Covance, respectively. DNA Constructs—Total RNA was extracted from HeLa cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentration was measured with a spectrophotometer, and RNA quality was determined by agarose gel electrophoresis. First strand cDNA synthesis was performed using 5 μg of total RNA and the avian myeloblastosis virus reverse transcriptase (Promega) with an oligo-dT primer. cDNAs were amplified using PRMT1 isoform-specific oligonucleotides (v1-7) designed to introduce the amplified fragments in frame with a hexahistidine tag in the EcoRI and XhoI sites of the pET-20b expression vector (Novagen). PRMT1v4 was amplified from an EST clone (IMAGE: 387210) using v4-specific primers, and PRMT1v7 was isolated using the v1- and v2-specific primers on HeLa cDNA. The resulting plasmids were digested with BamHI and XhoI, and the released full-length inserts were subsequently cloned in-frame with EGFP into the BglII and XhoI sites of the pEGFP-N1 vector (Clontech). The PRMT1v2-EGFP NES mutant, in which Val-15 and Leu-18 were replaced by alanines, was generated by using the Stratagene QuikChange mutagenesis kit. The pGFP-Rev-NES vector was generously provided by Dr. Stephen Lee (University of Ottawa, ON, Canada). The expression vectors encoding GST-fused RG motif of coilin (Val-385 to Val-423) and Sam68 (full-length or P3 (Gly-285 to Pro-308) proteins were kindly provided by Dr. Stephane Richard (McGill University). The expression vectors encoding GST-fused RG motifs of MRE11 (Phe-554 to Arg-680), SmB (Prp-206 to Leu-231), and SmB′ (Pro-206 to Pro-240) have been described (28Bedford M.T. Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Crossref PubMed Scopus (135) Google Scholar, 29Boisvert F.M. Dery U. Masson J.Y. Richard S. Genes Dev. 2005; 19: 671-676Crossref PubMed Scopus (166) Google Scholar). Full-length fibrillarin and hnRNP A1 were amplified from HeLa cDNA and introduced in-frame with a GST tag in the BamHI/XhoI restriction sites of the pGEX-4T2 vector (GE Healthcare). The DNA sequence of all constructs were verified by sequencing (StemCore Laboratories, Ottawa, ON, Canada). Purified ASF/SF2 protein was kindly provided by Dr. Martin Pelchat (University of Ottawa). RT-PCR Analysis—Total human tissue cDNAs were obtained from BioChain (Hayward, CA). PCR were performed in 25-μl reaction mixture using TaqDNA polymerase (Qiagen). cDNAs were amplified using the PF/PR primers (24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar) specific for PRMT1v1 to -3 or using either a forward PRMT1v4-specific primer (5′-aaatcttccagcggggtcgcg-3′), PRMT1v5-specific primer (5′-actggagagatggtgtcctgtgg-3′), PRMT1v6-specific primer (5′-aagctgaccagacaaagagagg-3′), or a PRMT1v7-specific primer (5′-tgcatcatggaggagatgctgaagg-3′) with the reverse PR primer. Primers specific for actin (24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar) were used to show that an equal amount of total cDNA was used for each reaction. An initial incubation at 98 °C for 2 min was followed by 35 cycles consisting of a 95 °C denaturation step (30 s), a 65 °C annealing step (30 s), and a 72 °C extension step (30 s). A final extension step at 72 °C was included for 10 min. For PRMT1 expression analysis in breast cancer cells, total RNA extraction and first strand cDNA synthesis were performed as described above. PCR reactions were also performed as described above but using 1 μl of a 1/10 cDNA dilution. Cycle number was determined empirically for each primer pair to stay within the linear range of amplification (see supplemental Fig. 1). All PCR products were electrophoresed on 2.5% agarose gels and visualized by ethidium bromide staining. RNA Silencing—The small interfering RNA (siRNA) specific to PRMT1 exon 2 was designed using Gene Link RNAi Explorer, and the siRNA-annealed oligonucleotide duplex was purchased from Invitrogen. Concomitantly, a PRMT1 exon 2-specific short hairpin RNA expression plasmid was generated according to Brummelkamp et al. (30Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar). Briefly, 60-bp synthetic DNA oligonucleotides containing PRMT1 exon 2-specific sense and corresponding antisense sequences flanking a 7-base hairpin were inserted into the BamHI and Hind III restriction sites of the pRS vector (OriGene). The siRNA duplex and the short hairpin RNA expression plasmid were concurrently transfected in HeLa cells using the X-tremeGENE siRNA transfection reagent (Roche Applied Science). Protein Purification—His6 fusion proteins were overexpressed in Escherichia coli BL-21 cells (Stratagene) by induction with a final concentration of 1 mm isopropyl-β-d-thiogalactopyranoside. After induction, cells were spun down, resuspended in 10 ml of lysis buffer (50 mm Na2HPO4, 300 mm NaCl, 10 mm imidazole, 20 mm 2-mercaptoethanol), and subsequently broken down by sonication (5 pulses of 15 s at 12 watts). Cell debris were discarded through centrifugation for 15 min at 10,000 × g. His6-tagged proteins were then purified using a nickel-nitrilotriacetic acid matrix (Qiagen) according to the manufacturer's instructions and eluted from the beads using 250 mm imidazole. GST fusion protein substrates (GST-coilin, -fibrillarin, -hnRNPA1, -MRE11, -Sam68, -SmB, and -SmB′) were expressed in E. coli BL-21 cells by induction with a final concentration of 0.1 mm isopropyl-β-d-thiogalactopyranoside. Cells were lysed by sonication as described above in 10 ml of 1× PBS supplemented with Complete protease inhibitor mixture (Roche Applied Science). The GST fusion proteins were purified using glutathione-agarose beads (Sigma) and after extensive washes with PBS wash buffer (1× PBS, 1% Triton X-100, Complete protease inhibitor) were eluted from the beads with 60 mm glutathione in PBS adjusted to pH 7.5. The purified tagged proteins were dialyzed against 1 liter of phosphate-buffered saline (1× PBS, 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4) at 4 °C overnight. The dialysates were then concentrated using Centricon centrifugal devices (Millipore). Endogenous protein substrates from PRMT1-/- embryonic stem (ES) cells were prepared as previously described (31Pawlak M.R. Banik-Maiti S. Pietenpol J.A. Ruley H.E. J. Cell. Biochem. 2002; 87: 394-407Crossref PubMed Scopus (37) Google Scholar). Briefly, cells were harvested in 50 mm sodium phosphate buffer, pH 7.5, and lysed by sonication. Cell debris were discarded through centrifugation for 10 min at 18,000 × g, and cell extracts were then heat-inactivated at 70 °C for 10 min. Alternatively, PRMT1-/- ES cells were fractionated using the QProteome nuclear protein kit (Qiagen) following manufacturer's instructions. All obtained fractions were then dialyzed against 50 mm sodium phosphate buffer, pH 7.5, at 4 °C for 16 h. Protein concentration was measured by using the DC protein assay reagent (Bio-Rad). Methylation Assays—In vitro methylation of the GST fusion proteins (5 μg) was performed at a final concentration of 25 mm Tris·HCl, pH 7.4. Methylation of the endogenous protein substrates from PRMT1-/- ES cells (15 μg of total proteins or 10 μg of each fraction) was performed in 50 mm sodium phosphate buffer, pH 7.5. All methylation reactions were performed in a final volume of 30 μl and contained 1 μg (∼0.8 μm final) of one of the purified His6-tagged PRMT1 protein isoforms v1 to v7 and a final concentration of 0.582 μm S-adenosyl-l-[methyl-3H]methionine (PerkinElmer Life Sciences; 63.6 Ci/mmol, from a stock in 10 mm sulfuric acid solution:ethanol (9:1) at a concentration of 0.55 mCi/ml), except for the reactions showed in Fig. 2B, where 50 μm concentrations of cold S-adenosyl-l-methionine was added to the reaction. Methylation reactions were allowed to proceed at 37 °C for 2 h. Methylated proteins were then resolved by SDS-PAGE. After electrophoresis, gels were stained in Coomassie Brilliant Blue R-250, destained in a 10% methanol (v/v), 5% acetic acid (v/v) destaining solution to visualize protein bands, and then soaked in En3Hance (PerkinElmer Life Sciences) according to the manufacturer's instructions. Gels were dried in vacuo, and 3H-labeled proteins were visualized by fluorography. All fluorographs were exposed at -80 °C for 1 week. Mean methylation activity ±S.E. for each isoform-substrate pair is expressed as an arbitrary unit derived from the band intensities on the fluorograph, normalized to the amount of isoform and substrate seen on the Coomassie stain. These intensity values were obtained using the Gel Plotting macro of ImageJ v1.34p. Each methylation assay has been reproduced at least three times, and one-way analysis of variance statistical analysis was performed to verify that differences in mean relative activities were significant (p < 0.05; 95% confidence intervals). Genomic Structure of the Human Prmt1 Gene—Upon initial identification of the Prmt1 genomic locus, it was observed that three transcripts could be produced through alternative splicing of exons 2 and 3 (named v1-v3; Fig. 1A), and expression of these variants at the RNA level was later confirmed by an independent study (23Scott 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, 24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar). Zhang and Cheng (32Zhang X. Cheng X. Structure. 2003; 11: 509-520Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) then predicted the existence of three additional isoforms (named v4-v6; Fig. 1A) by comparing available ESTs with human PRMT1 genomic sequence but did not investigate further how these mRNAs were produced. Mapping of the unique sequences present at the 5′-end of v4 and v6 mRNAs onto the human PRMT1 genomic locus (human Mar. 2006 hg18 assembly; NCBI Build 36.1) using the UCSC Genome Bioinformatics BLAT alignment tool (33Kent W.J. Genome Res. 2002; 12: 656-664Crossref PubMed Scopus (6161) Google Scholar), suggested the existence of at least two additional exons upstream of the previously identified first exon. Further examination of splicing junctions in the genomic sequence using UCSC Genome Browser (34Kent W.J. Sugnet C.W. Furey T.S. Roskin K.M. Pringle T.H. Zahler A.M. Haussler D. Genome Res. 2002; 12: 996-1006Crossref PubMed Scopus (6719) Google Scholar) revealed the presence of four alternative 5′-exons in the Prmt1 gene that we have renamed e1a-d, e1d being the exon previously labeled exon 1 (Fig. 1A). e1c and e1d do not seem to have functional 3′ splice sites (3′ss) and, hence, are never joined together and are utilized as alternate first exons. In contrast, exon 1b has two alternative 3′ss that can be paired with the 5′ss of e1a. In addition, the intron between these exons is sometimes retained in the mRNA (Fig. 1A). These splicing events would take place ∼1 kilobase away from the previously identified exon 1 and support the presence of a transcriptional start site (TSS1) upstream of e1a. Indeed, a promoter (genomic coordinates 54,870,654-54,870,904) and a putative TSS (54,871,225) are predicted in this region using the “Promoter Scan” and “DBTSS” algorithms (35Knudsen S. Bioinformatics. 1999; 15: 356-361Crossref PubMed Scopus (275) Google Scholar, 36Suzuki Y. Yamashita R. Nakai K. Sugano S. Nucleic Acids Res. 2002; 30: 328-331Crossref PubMed Scopus (173) Google Scholar). However, the highest score obtained for a TSS using the Neural Network Promoter Prediction algorithm (37Reese M.G. Comput. Chem. 2001; 26: 51-56Crossref PubMed Scopus (691) Google Scholar) maps to a site just upstream of e1d (54,872,277), which is compatible with this exon being the most represented first exon among ESTs. Preliminary results from a 5′-rapid amplification of cDNA ends analysis of PRMT1 mRNAs confirms the existence of this TSS2 (data not shown and Fig. 1A). Thus, these observations are consistent with a model where alternative promoters would dictate at least in part the choice of 5′-exon usage. The splicing patterns of alternative exons 2, 3, 4, and 5 are also more complex than initially anticipated from previous reports (23Scott 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, 24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar) and involve the use of multiple alternative 5′ss and 3′ss. For example, the intron between e2 and e3 includes minor spliceosome AT-AC splice sites (38Tarn W.Y. Steitz J.A. Trends Biochem. Sci. 1997; 22: 132-137Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 39Tarn W.Y. Steitz J.A. Cell. 1996; 84: 801-811Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 40Hall S.L. Padgett R.A. Science. 1996; 271: 1716-1718Crossref PubMed Scopus (171) Google Scholar, 41Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (186) Google Scholar) in addition to a non-canonical GT-cc pair. In this case the 5′ss used determines the reading frame irrespective of the 3′ss to which it is paired and, thus, will dictate AUG start codon choice; GT results in the use of a start codon in e3 and AT in e4, yielding protein isoforms v3 and v6, respectively. Finally, protein isoform v5 results from pairing of e1d-e2-e3 (GT-cc) but with usage of an alternative 5′ss downstream of e3 (Fig. 1A). The predicted protein-coding regions (excluding 5′ and 3′-untranslated regions) of the various Prmt1 transcripts are composed of 1059 nt (v1), 1113 nt (v2), 1041 nt (v3), 1047 nt (v4), 1026 nt (v5), 975 nt (v6), or 960 nt (v7) and would encode 7 deduced polypeptides with a predicted molecular mass of 40.5, 42.5, 39.9, 40.1, 39.4, 37.7, and 36.7 kDa, respectively, excluding any posttranslational modifications (Fig. 1A). Expression Profile of PRMT1 Isoforms in Normal Human Tissues—Experiments investigating the expression pattern of PRMT1v1, -v2, and -v3 in different human tissues have demonstrated that these variants are ubiquitously expressed (23Scott 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, 24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar). To address the expression profile of the newly isolated isoforms, we analyzed the relative expression levels of the seven PRMT1 isoforms in human normal tissues by semiquantitative RT-PCR using the expression level of the actin gene as an internal control (Fig. 1B). PRMT1v1 was mostly expressed in the kidney, liver, lung, skeletal muscle, and spleen, whereas PRMT1v2 was predominantly detectable in the kidney, liver, and pancreas. The PRMT1v3 isoform was present at comparable, but low levels in all tested tissues. In contrast, PRMT1v4 to -7 showed a higher degree of tissue specificity in their expression patterns. For example, v4 was detectable only in the heart, whereas v5 was mostly expressed in the pancreas, and v7 was predominantly present in the heart and skeletal muscles (Fig. 1B). Finally, the predicted PRMT1v6 mRNA was not detectable in any normal tissues tested under the experimental conditions used here. These results show that the expression level of the PRMT1 variants varies significantly between human tissues. PRMT1 Expression in Breast Cancer Cells—It has been previously reported that the relative prevalence of PRMT1 isoforms 1-3 is different between normal and cancerous breast tissues (24Scorilas A. Black M.H. Talieri M. Diamandis E.P. Biochem. Biophys. Res. Commun. 2000; 278: 349-359Crossref PubMed Scopus (59) Google Scholar). In addition, a large proportion of ESTs for PRMT1v6 were obtained from cancer cells libraries, suggesting that the balance of specific alternative isoforms might be altered in transformed cells. To assess whether the expression of PRMT1 N-terminal variants is distinct in breast cancer cells, we examined the expression of the Prmt1 gene in normal and cancerous breast cell lines using semiquantitative RT-PCR. Actin mRNA was used as a control for the amount of cDNA used (Fig. 1C, bottom panel, and supplemental Fig. 1). We find that PRMT1 expression level is on average 14-fold higher among the breast cancer cell lines tested (Fig. 1C). Furthermore, the relative expression ratio of the isoforms also varied between normal and breast cancer cell lines. Specifically, the ratio of v2 over v1 mRNAs was increased on average 3.5-fold in breast cancer cell lines (Fig. 1F), suggesting that the v2 isoform is selectively increased relative to v1. Among the other isoforms, PRMT1v5 and -v6 were detected uniquely in specific breast cancer cell lines but not in normal breast cells (Fig. 1C, compare lanes 3, 6, and 7, with lane 1). The newly identified v7 isoform expression level was also on average 3-fold higher in the breast cancer lines te