Publications

• The novel human protein arginine N-methyltransferase PRMT6 is a nuclear enzyme displaying unique substrate specificity.

  Frankel A., Yadav N., Lee J., Branscombe T.L., Clarke S., Bedford M.T.

  Protein arginine methylation is a prevalent posttranslational modification in eukaryotic cells that has been implicated in signal transduction, the metabolism of nascent pre-RNA, and the transcriptional activation processes. In searching the human genome for protein arginine N-methyltransferase (P ... >> More

  Protein arginine methylation is a prevalent posttranslational modification in eukaryotic cells that has been implicated in signal transduction, the metabolism of nascent pre-RNA, and the transcriptional activation processes. In searching the human genome for protein arginine N-methyltransferase (PRMT) family members, a novel gene has been found on chromosome 1 that encodes for an apparent methyltransferase, PRMT6. The polypeptide chain of PRMT6 is 41.9 kDa consisting of a catalytic core sequence common to other PRMT enzymes. Expressed as a glutathione S-transferase fusion protein, PRMT6 demonstrates type I PRMT activity, capable of forming both omega-N(G)-monomethylarginine and asymmetric omega-N(G),N(G)-dimethylarginine derivatives on the recombinant glycine- and arginine-rich substrate in a processive manner with a specific activity of 144 pmol methyl groups transferred min(-1) mg(-1) enzyme. A comparison of substrate specificity reveals that PRMT6 is functionally distinct from two previously characterized type I enzymes, PRMT1 and PRMT4. In addition, PRMT6 displays automethylation activity; it is the first PRMT to do so. This novel human PRMT, which resides solely in the nucleus when fused to the green fluorescent protein, joins a family of enzymes with diverse functions within cells. << Less

  J. Biol. Chem. 277:3537-3543(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Unique features of human protein arginine methyltransferase 9 (PRMT9) and its substrate RNA splicing factor SF3B2.

  Hadjikyriacou A., Yang Y., Espejo A., Bedford M.T., Clarke S.G.

  Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed e ... >> More

  Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. << Less

  J. Biol. Chem. 290:16723-16743(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• RNA and protein interactions modulated by protein arginine methylation.

  Gary J.D., Clarke S.

  This review summarizes the current status of protein arginine N-methylation reactions. These covalent modifications of proteins are now recognized in a number of eukaryotic proteins and their functional significance is beginning to be understood. Genes that encode those methyltransferases specific ... >> More

  This review summarizes the current status of protein arginine N-methylation reactions. These covalent modifications of proteins are now recognized in a number of eukaryotic proteins and their functional significance is beginning to be understood. Genes that encode those methyltransferases specific for catalyzing the formation of asymmetric dimethylarginine have been identified. The enzyme modifies a number of generally nuclear or nucleolar proteins that interact with nucleic acids, particularly RNA. Postulated roles for these reactions include signal transduction, nuclear transport, or a direct modulation of nucleic acid interactions. A second methyltransferase activity that symmetrically dimethylates an arginine residue in myelin basic protein, a major component of the axon sheath, has also been characterized. However, a gene encoding this activity has not been identified to date and the cellular function for this methylation reaction has not been clearly established. From the analysis of the sequences surrounding known arginine methylation sites, we have determined consensus methyl-accepting sequences that may be useful in identifying novel substrates for these enzymes and may shed further light on their physiological role. << Less

  Prog Nucleic Acid Res Mol Biol 61:65-131(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• PRMT 3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation.

  Tang J., Gary J.D., Clarke S., Herschman H.R.

  Methylation is one of the many post-translational modifications that modulate protein function. Although asymmetric NG,NG-dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine N-methyltransferases (PRMT), has been known for ... >> More

  Methylation is one of the many post-translational modifications that modulate protein function. Although asymmetric NG,NG-dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine N-methyltransferases (PRMT), has been known for some time, members of this enzyme class have only recently been cloned. The first example of this type of enzyme, designated PRMT1, cloned because of its ability to interact with the mammalian TIS21 immediate-early protein, was then shown to have protein arginine methyltransferase activity. We have now isolated rat and human cDNA orthologues that encode proteins with substantial sequence similarity to PRMT1. A recombinant glutathione S-transferase (GST) fusion product of this new rat protein, named PRMT3, asymmetrically dimethylates arginine residues present both in the designed substrate GST-GAR and in substrate proteins present in hypomethylated extracts of a yeast rmt1 mutant that lacks type I arginine methyltransferase activity; PRMT3 is thus a functional type I protein arginine N-methyltransferase. However, rat PRMT1 and PRMT3 glutathione S-transferase fusion proteins have distinct enzyme specificities for substrates present in both hypomethylated rmt1 yeast extract and hypomethylated RAT1 embryo cell extract. TIS21 protein modulates the enzymatic activity of recombinant GST-PRMT1 fusion protein but not the activity of GST-PRMT3. Western blot analysis of gel filtration fractions suggests that PRMT3 is present as a monomer in RAT1 cell extracts. In contrast, PRMT1 is present in an oligomeric complex. Immunofluorescence analysis localized PRMT1 predominantly to the nucleus of RAT1 cells. In contrast, PRMT3 is predominantly cytoplasmic. << Less

  J. Biol. Chem. 273:16935-16945(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.

  Gonsalvez G.B., Tian L., Ospina J.K., Boisvert F.-M., Lamond A.I., Matera A.G.

  Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal ... >> More

  Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. << Less

  J. Cell Biol. 178:733-740(2007) [PubMed] [EuropePMC]

• PRMT5 (Janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins.

  Branscombe T.L., Frankel A., Lee J.H., Cook J.R., Yang Z., Pestka S., Clarke S.

  We have identified a new mammalian protein arginine N-methyltransferase, PRMT5, formerly designated Janus kinase-binding protein 1, that can catalyze the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine in a variety of proteins. A hemagglutinin peptide-tag ... >> More

  We have identified a new mammalian protein arginine N-methyltransferase, PRMT5, formerly designated Janus kinase-binding protein 1, that can catalyze the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine in a variety of proteins. A hemagglutinin peptide-tagged PRMT5 complex purified from human HeLa cells catalyzes the S-adenosyl-l-[methyl-(3)H]methionine-dependent in vitro methylation of myelin basic protein. When the radiolabeled myelin basic protein was acid-hydrolyzed to free amino acids, and the products were separated by high-resolution cation exchange chromatography, we were able to detect two tritiated species. One species co-migrated with a omega-N(G)-monomethylarginine standard, and the other co-chromatographed with a symmetric omega-N(G),N(G')-dimethylarginine standard. Upon base treatment, this second species formed methylamine, a breakdown product characteristic of symmetric omega-N(G),N(G')-dimethylarginine. Further analysis of these two species by thin layer chromatography confirmed their identification as omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine. The hemagglutinin-PRMT5 complex was also able to monomethylate and symmetrically dimethylate bovine histone H2A and a glutathione S-transferase-fibrillarin (amino acids 1-148) fusion protein (glutathione S-transferase-GAR). A mutation introduced into the S-adenosyl-l-methionine-binding motif I of a myc-tagged PRMT5 construct in COS-1 cells led to a near complete loss of observed enzymatic activity. PRMT5 is the first example of a catalytic chain for a type II protein arginine N-methyltransferase that can result in the formation of symmetric dimethylarginine residues as observed previously in myelin basic protein, Sm small nuclear ribonucleoproteins, and other polypeptides. << Less

  J Biol Chem 276:32971-32976(2001) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Substrate specificity of human protein arginine methyltransferase 7 (PRMT7): the importance of acidic residues in the double E loop.

  Feng Y., Hadjikyriacou A., Clarke S.G.

  Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however ... >> More

  Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. << Less

  J. Biol. Chem. 289:32604-32616(2014) [PubMed] [EuropePMC]

• PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells.

  Tang J., Frankel A., Cook R.J., Kim S., Paik W.K., Williams K.R., Clarke S., Herschman H.R.

  Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity ... >> More

  Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues. << Less

  J Biol Chem 275:7723-7730(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and characterization of enzymes from Euglena gracilis that methylate methionine and arginine residues of cytochrome c.

  Farooqui J.Z., Tuck M., Paik W.K.

  Two forms of cytochrome c-specific methyltransferases from Euglena gracilis were purified approximately 100- and 50-fold, respectively, using DEAE-cellulose and gel-filtration chromatography. The methylation product of enzyme I was identified as S-methylmethionine and that of enzyme II as NG-monom ... >> More

  Two forms of cytochrome c-specific methyltransferases from Euglena gracilis were purified approximately 100- and 50-fold, respectively, using DEAE-cellulose and gel-filtration chromatography. The methylation product of enzyme I was identified as S-methylmethionine and that of enzyme II as NG-monomethylarginine. Both enzymes were located in the cytosol and exhibit maximum activity at pH 7.0. Among the various proteins tested as substrates, the enzymes were highly specific toward cytochrome c. Various types of histones, in particular, were not modified by either enzyme. The molecular weights of enzyme I and II were 28,000 and 36,000, respectively. Various S-adenosyl-L-homocysteine analogs were tested for their inhibitory activity toward the enzymes. Only the D- and L-isomers of S-adenosylhomocysteine and sinefungin were significantly inhibitory. The Ki values for S-adenosyl-L-homocysteine were 8.13 X 10(-6) and 1.17 X 10(-5) M for enzyme I and II, respectively. Two-dimensional peptide mapping revealed the methylation site of enzyme I to be the methionine residue at position 65 while that of enzyme II to be the arginine residue at position 38. The methylation of either methionine or arginine residues by enzyme I and II, respectively, lowers the isoelectric point (pI) of cytochrome c: 9.23, 9.33, and 10.06 for S-methylmethionine-, NG-monomethylarginine-modified, and unmodified cytochrome c, respectively. << Less

  J Biol Chem 260:537-545(1985) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity.

  Miranda T.B., Miranda M., Frankel A., Clarke S.

  We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli ... >> More

  We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-3H]-l-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an omega-NG-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1-148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality. << Less

  J. Biol. Chem. 279:22902-22907(2004) [PubMed] [EuropePMC]

• Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme.

  Ghosh S.K., Paik W.K., Kim S.

  Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylam ... >> More

  Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylamide gel electrophoresis. Although both methylases share some common properties, such as utilization of S-adenosyl-L-methionine as the methyl donor and methylation of protein-bound arginine residues, they are distinctly different from each other in molecular weight and in catalytic, as well as the immunological, properties. The MBP-specific protein methylase I (approximately 500 kDa) methylates MBP preferentially (Km = 2 X 10(-7) M) and histone to a much lesser extent (Km = 1 X 10(-4) M), while the histone-specific methylase I (approximately 275 kDa) methylates histone only. Both methylases exhibit two major subunit bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 100 and 72 kDa for the MBP-specific and 110 and 75 kDa for the histone-specific. At 0.5 mM p-chloromercuribenzoate, about 50% of the MBP-specific enzyme remained as active, while most of the histone-specific enzyme activity was lost. In 2 mM guanidine HCl, approximately 90% of the former enzyme activity remained while nearly complete inactivation of the latter enzyme was observed. The enzymes also exhibited quite different inactivation profiles toward high temperature (45-65 degrees C); MBP-enzyme was stable up to 50 degrees C and was rapidly inactivated at higher temperatures with an inflection point at about 57 degrees C. However, under the identical conditions, histone-enzyme was inactivated progressively and linearly in the same temperature range. Finally, Western immunoblot analysis of polyclonal antibodies directed against either enzyme exhibited no cross-reactivity with the other. << Less

  J Biol Chem 263:19024-19033(1988) [PubMed] [EuropePMC]