The development and characterization of a chemical probe targeting PRMT1 over PRMT5

Protein arginine methyltransferases (PRMTs) are a family of mammalian enzymes catalyzing the symmetric dimethylation (Type I), asymmetric dimethylation (Type II), or monomethylation (Type III) of arginine residues within proteins. This family is composed of 11 isozymes, however the vast majority of asymmetric and symmetric dimethylation in mammals is completed by either PRMT1 or PRMT5, respectively. In recent years, a number of chemical probes targeting this family of enzymes have been developed, but the majority of these probes lack isozyme specificity. Herein, we report the development of a chemical probe, based on a non-natural peptide sequence, which specifically labels PRMT1 over PRMT5 with high selectivity and sensitivity.     

PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.     

FBXO11/PRMT9, a new protein arginine methyltransferase, symmetrically dimethylates arginine residues

We have identified a protein, FLJ12673 or FBXO11, that contains domains characteristically present in protein arginine methyltransferases (PRMTs). Immuno-purified protein expressed from one of the four splice variants in HeLa cells and in Escherichia coli exhibited methyltransferase activity. Monomethylarginine, symmetric, and asymmetric dimethylarginine (SDMA, ADMA) were formed on arginine residues. Accordingly, we have designated the protein PRMT9. PRMT9 is the third member of the PRMT family that forms SDMA modifications in proteins. Structurally, this protein is distinct from all other known PRMTs implying that convergent evolution allowed this protein to develop the ability to methylate arginine residues and evolved elements conserved in PRMTs to accomplish this.     

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2

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.     

Understanding protein arginine methyltransferase 1 (PRMT1) product specificity from molecular dynamics

Protein arginine methyltransferases (PRMTs) catalyze the post-translational methylation of specific arginyl groups within targeted proteins to regulate fundamental biological responses in eukaryotic cells. The major Type I PRMT enzyme, PRMT1, strictly generates monomethyl arginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). Multiple diseases can arise from the dysregulation of PRMT1, including heart disease and cancer, which underscores the need to elucidate the origin of product specificity. Molecular dynamics (MD) simulations were carried out for WT PRMT1 and its M48F, H293A, H293S, and H293S-M48F mutants bound with S-adenosylmethionine (AdoMet) and the arginine substrate in an unmethylated or methylated form. Experimental site-directed mutagenesis and analysis of the resultant products were also performed. Two specific PRMT1 active site residues, Met48 and His293, have been determined to play a key role in dictating product specificity, as: (1) the single mutation of Met48 to Phe enabled PRMT1 to generate MMA, ADMA, and a limited amount of SDMA; (2) the single mutation of His293 to Ser formed the expected MMA and ADMA products only; whereas (3) the double mutant H293S-M48F-PRMT1 produced SMDA as the major product with limited amounts of MMA and ADMA. Calculating the formation of near-attack conformers resembling S_N2 transition states leading to either the ADMA or SDMA products finds that Met48 and His293 may enable WT PRMT1 to yield ADMA exclusively by precluding MMA from binding in an orientation more conducive to SDMA formation, i.e., the methyl group bound at the arginine N_η2 position.     

PRMT5 promotes colorectal cancer growth by interaction with MCM7

Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse arginine methylation of histones and non-histone proteins. Accumulating evidence indicates that PRMT5 promotes cancer development and progression. However, its function in colorectal cancer (CRC) is poorly understood. In this study, we revealed the oncogenic roles of PRMT5 in CRC. We found that PRMT5 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. We identified minichromosome maintenance-7 (MCM7) as the direct PRMT5-binding partner. A co-immunoprecipitation (co-IP) assay indicated that PRMT5 physically interacted with MCM7 and that the direct binding domain was located between residues 1-248 in MCM7. In addition, our results from analysis of 99 CRC tissues and 77 adjacent non-cancerous tissues indicated that the PRMT5 and MCM7 expression levels were significantly higher in CRC tissues than in control tissues, which was further confirmed by bioinformatic analysis using TCGA and GEO datasets. We also found that MCM7 promoted CRC cell proliferation, migration and invasion in vitro. Furthermore, we observed that increased PRMT5 expression predicted unfavourable patient survival in CRC patients and in the subgroup of patients with a tumour size of ≤5 cm. These data suggested that PRMT5 and MCM7 might be novel potential targets for the treatment of CRC.     

PRMT5 determines the pattern of polyploidization and prevents liver from cirrhosis and carcinogenesis

Human hepatocellular carcinoma(HCC) occurs almost exclusively in cirrhotic livers. Here, we report that hepatic loss of protein arginine methyltransferase 5(PRMT5) in mice is sufficient to cause cirrhosis and HCC in a clinically relevant way. Furthermore, pathological polyploidization induced by hepatic loss of PRMT5 promotes liver cirrhosis and hepatic tumorigenesis in aged liver. The loss of PRMT5 leads to hyperaccumulation of P21 and endoreplication-dependent formation of pathological mono-nu...     

Kinetic mechanism of protein arginine methyltransferase 1

Protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and dimethylation of peptidyl arginine residues. Although all PRMTs produce monomethyl arginine (MMA), type 1 PRMTs go on to form asymmetrically dimethylated arginine (ADMA), while type 2 enzymes form symmetrically dimethylated arginine (SDMA). PRMT1 is the major type 1 PRMT in vivo, thus it is the primary producer of the competitive NOS inhibitor, ADMA. Hence, potent inhibitors, which are highly selective for this particular isozyme, could serve as excellent therapeutics for heart disease. However, the design of such inhibitors is impeded by a lack of information regarding this enzyme's kinetic and catalytic mechanisms. Herein we report an analysis of the kinetic mechanism of human PRMT1 using both an unmethylated and a monomethylated substrate peptide based on the N-terminus of histone H4. The results of initial velocity and product and dead-end inhibition experiments indicate that PRMT1 utilizes a rapid equilibrium random mechanism with the formation of dead-end EAP and EBQ complexes. This mechanism is gratifyingly consistent with previous results demonstrating that PRMT1 catalyzes substrate dimethylation in a partially processive manner.     

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.     

Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3

Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain NG, NG-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.     

Type II arginine methyltransferase PRMT5 regulates gene expression of inhibitors of differentiation/DNA binding Id2 and Id4 during glial cell differentiation

PRMT5 is a type II protein arginine methyltranferase that catalyzes monomethylation and symmetric dimethylation of arginine residues. PRMT5 is functionally involved in a variety of biological processes including embryo development and circadian clock regulation. However, the role of PRMT5 in oligodendrocyte differentiation and central nervous system myelination is unknown. Here we show that PRMT5 expression gradually increases throughout postnatal brain development, coinciding with the period of active myelination. PRMT5 expression was observed in neurons, astrocytes, and oligodendrocytes. siRNA-mediated depletion of PRMT5 in mouse primary oligodendrocyte progenitor cells abrogated oligodendrocyte differentiation. In addition, the PRMT5-depleted oligodendrocyte progenitor and C6 glioma cells expressed high levels of the inhibitors of differentiation/DNA binding, Id2 and Id4, known repressors of glial cell differentiation. We observed that CpG-rich islands within the Id2 and Id4 genes were bound by PRMT5 and were hypomethylated in PRMT5-deficient cells, suggesting that PRMT5 plays a role in gene silencing during glial cell differentiation. Our findings define a role of PRMT5 in glial cell differentiation and link PRMT5 to epigenetic changes during oligodendrocyte differentiation.     

RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity

Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins.     

Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

Protein arginine methyltransferase 5 (PRMT5) is a key epigenetic regulator that symmetrically dimethylates arginine residues on histones H3 and H4 to silence gene expression. PRMT5 is frequently observed in a complex with the cofactor methylosome protein 50 (MEP50), which is required for PRMT5 activity. PKCδ/p38δ signaling, a key controller of keratinocyte proliferation and differentiation, increases p21^Cip1 expression to suppress keratinocyte proliferation. We now show that MEP50 enhances keratinocyte proliferation and survival via mechanisms that include silencing of p21^Cip1 expression. This is associated with enhanced PRMT5-MEP50 interaction at the p21^Cip1 promoter and enhanced arginine dimethylation of the promoter-associated histones H3 and H4. It is also associated with a MEP50-dependent reduction in the level of p53, a key controller of p21^Cip1 gene expression. We confirm an important biological role for MEP50 and PRMT5 in regulating keratinocyte proliferation using a stratified epidermal equivalent model that mimics in vivo epidermal keratinocyte differentiation. In this model, PRMT5 or MEP50 knockdown results in reduced keratinocyte proliferation. We further show that PKCδ/p38δ signaling suppresses MEP50 expression, leading to reduced H3/H4 arginine dimethylation at the p21^Cip1 promoter, and that this is associated with enhanced p21^Cip1 expression and reduced cell proliferation. These findings describe an opposing action between PKCδ/p38δ MAPK signaling and PRMT5/MEP50 epigenetic silencing mechanisms in regulating cell proliferation.     

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.