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

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.     

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

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.     

Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues

Full-length human protein arginine methyltransferase 7 (PRMT7) expressed as a fusion protein in Escherichia coli was initially found to generate only ω-N(G)-monomethylated arginine residues in small peptides, suggesting that it is a type III enzyme. A later study, however, characterized fusion proteins of PRMT7 expressed in bacterial and mammalian cells as a type II/type I enzyme, capable of producing symmetrically dimethylated arginine (type II activity) as well as small amounts of asymmetric dimethylarginine (type I activity). We have sought to clarify the enzymatic activity of human PRMT7. We analyzed the in vitro methylation products of a glutathione S-transferase (GST)-PRMT7 fusion protein with robust activity using a variety of arginine-containing synthetic peptides and protein substrates, including a GST fusion with the N-terminal domain of fibrillarin (GST-GAR), myelin basic protein, and recombinant human histones H2A, H2B, H3, and H4. Regardless of the methylation reaction conditions (incubation time, reaction volume, and substrate concentration), we found that PRMT7 only produces ω-N(G)-monomethylarginine with these substrates. In control experiments, we showed that mammalian GST-PRMT1 and Myc-PRMT5 were, unlike PRMT7, able to dimethylate both peptide P-SmD3 and SmB/D3 to give the expected asymmetric and symmetric products, respectively. These experiments show that PRMT7 is indeed a type III human methyltransferase capable of forming only ω-N(G)-monomethylarginine, not asymmetric ω-N(G),N(G)-dimethylarginine or symmetric ω-N(G),N(G')-dimethylarginine, under the conditions tested.     

A kinetic study of human protein arginine N-methyltransferase 6 reveals a distributive mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.     

Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A

The HSL7 (histone synthetic lethal 7) gene in the yeast Saccharomyces cerevisiae encodes a protein with close sequence similarity to the mammalian PRMT5 protein, a member of the class of protein arginine methyltransferases that catalyses the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N'(G)-dimethylarginine residues in a number of methyl-accepting species. A full-length HSL7 construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hsl7 effectively catalyses the transfer of methyl groups from S-adenosyl-[methyl-3H]-L-methionine to calf thymus histone H2A. When the acid-hydrolysed radiolabelled protein products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species that co-migrated with an omega-N(G)-monomethylarginine standard. No radioactivity was observed that co-migrated with either the asymmetric or symmetric dimethylated derivatives. In control experiments, no methylation of histone H2A was found with two mutant constructs of Hsl7. Surprisingly, FLAG-Hsl7 does not appear to effectively catalyse the in vitro methylation of a GST (glutathione S-transferase)-GAR [glycine- and arginine-rich human fibrillarin-(1-148) peptide] fusion protein or bovine brain myelin basic protein, both good methyl-accepting substrates for the human homologue PRMT5. Additionally, FLAG-Hsl7 demonstrates no activity on purified calf thymus histones H1, H2B, H3 or H4. GST-Rmt1, the GST-fusion protein of the major yeast protein arginine methyltransferase, was also found to methylate calf thymus histone H2A. Although we detected Rmt1-dependent arginine methylation in vivo in purified yeast histones H2A, H2B, H3 and H4, we found no evidence for Hsl7-dependent methylation of endogenous yeast histones. The physiological substrates of the Hsl7 enzyme remain to be identified.     

PRMT3 is a ribosomal protein methyltransferase that affects the cellular levels of ribosomal subunits

The mammalian protein arginine methyltransferase 3 (PRMT3) catalyzes the formation of asymmetric (type I) dimethylarginine in vitro. As yet, natural substrates and cellular pathways modulated by PRMT3 remain unknown. Here, we have identified an ortholog of PRMT3 in fission yeast. Tandem affinity purification of fission yeast PRMT3 coupled with mass spectrometric protein identification revealed that PRMT3 associates with components of the translational machinery. We identified the 40S ribosomal protein S2 as the first physiological substrate of PRMT3. In addition, a fraction of yeast and human PRMT3 cosedimented with free 40S ribosomal subunits, as determined by sucrose gradient velocity centrifugation. The activity of PRMT3 is not essential since prmt3-disrupted cells are viable. Interestingly, cells lacking PRMT3 showed an accumulation of free 60S ribosomal subunits resulting in an imbalance in the 40S:60S free subunits ratio; yet pre-rRNA processing appeared to occur normally. Our results identify PRMT3 as the first type I ribosomal protein arginine methyltransferase and suggest that it regulates ribosome biosynthesis at a stage beyond pre-rRNA processing.     

Crystal structure of the plant epigenetic protein arginine methyltransferase 10

Protein arginine methyltransferase 10 (PRMT10) is a type I arginine methyltransferase that is essential for regulating flowering time in Arabidopsis thaliana. We present a 2.6 Å resolution crystal structure of A. thaliana PRMT 10 (AtPRMT10) in complex with a reaction product, S-adenosylhomocysteine. The structure reveals a dimerization arm that is 12-20 residues longer than PRMT structures elucidated previously; as a result, the essential AtPRMT10 dimer exhibits a large central cavity and a distinctly accessible active site. We employ molecular dynamics to examine how dimerization facilitates AtPRMT10 motions necessary for activity, and we show that these motions are conserved in other PRMT enzymes. Finally, functional data reveal that the 10 N-terminal residues of AtPRMT10 influence substrate specificity, and that enzyme activity is dependent on substrate protein sequences distal from the methylation site. Taken together, these data provide insights into the molecular mechanism of AtPRMT10, as well as other members of the PRMT family of enzymes. They highlight differences between AtPRMT10 and other PRMTs but also indicate that motions are a conserved element of PRMT function.     

Cell Cycle Regulation by the PRMT6 Arginine Methyltransferase through Repression of Cyclin-Dependent Kinase Inhibitors

PRMT6 belongs to the family of Protein Arginine Methyltransferase (PRMT) enzymes that catalyze the methylation of guanidino nitrogens of arginine residues. PRMT6 has been shown to modify the tail of histone H3, but the in vivo function of PRMT6 is largely unknown. Here, we show that PRMT6 regulates cell cycle progression. Knockdown of PRMT6 expression in the human osteosarcoma cell line U2OS results in an accumulation of cells at the G2 checkpoint. Loss of PRMT6 coincides with upregulation of p21 and p27, two members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors. Gene expression and promoter analysis show that p21 and p27 are direct targets of PRMT6, which involves methylation of arginine-2 of histone H3. Our findings imply arginine methylation of histones by PRMT6 in cell cycle regulation.     

Peptide backbone conformation affects the substrate preference of protein arginine methyltransferase I

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.     

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.     

Down-regulation of asymmetric arginine methylation during replicative and H2O2-induced premature senescence in WI-38 human diploid fibroblasts

Protein arginine methylation is one of the post-translational modifications which yield monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. In the present study, we investigated the status of protein arginine methylation during human diploid fibroblast senescence. When the expression of protein arginine methyltransferases (PRMTs), namely PRMT1, PRMT4, PRMT5 and PRMT6 was examined, a significant reduction was found in replicatively senescent cells as well as their catalytic activities against histone mixtures compared with the young cells. Furthermore, when the endogenous level of arginine-dimethylated proteins was determined, asymmetric modification (the product of type I PRMTs including PRMT1, PRMT4 and PRMT6) was markedly down-regulated. In contrast, both up- and down-regulations of symmetrically arginine-methylated proteins (the product of type II PRMTs including PRMT5) during replicative senescence were found. Furthermore, when young fibroblasts were induced to premature senescence by sub-cytotoxic H2O2 treatment, results similar to replicative senescence were obtained. Finally, we found that SV40-mediated immortalized WI-38 and HeLa cell lines maintained a higher level of asymmetrically modified proteins as well as type I PRMTs than young fibroblasts. These results suggest that the maintenance of asymmetric modification in the expressed target proteins of type I PRMTs might be critical for cellular proliferation.