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.     

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...     

A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high‐resolution mass spectrometry, a total of 147 symmetric dimethyl‐arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH‐type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.     

Kinetic mechanism of protein arginine methyltransferase 6 (PRMT6)

The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the mono- and dimethylation of arginine residues in a variety of proteins. Although these enzymes play important roles in a variety of cellular processes, aberrant PRMT activity is associated with several disease states, including heart disease and cancer. In an effort to guide the development of inhibitors targeting individual PRMTs, we initiated studies to characterize the molecular mechanisms of PRMT catalysis. Herein, we report studies on the kinetic mechanism of PRMT6. Initial velocity, product inhibition, and dead-end analog inhibition studies with the AcH4-21 and R1 peptides, as well as their monomethylated versions, indicate, in contrast to a previous report, that PRMT6 utilizes a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes.     

PRMT5 regulates cell pyroptosis by silencing CASP1 in multiple myeloma

Protein arginine methyltransferase 5 (PRMT5), a histone methyltransferase responsible for the symmetric dimethylation of histone H4 on Arg 3 (H4R3me2s), is an enzyme that participates in tumor cell progression in a variety of hematological malignancies. However, the biological functions of PRMT5 in multiple myeloma (MM) and the underlying molecular mechanisms remain unclear. In this study, we conducted a bioinformatics analysis and found that PRMT5 expression was significantly upregulated in MM. In vitro and in vivo phenotypic experiments revealed that knockdown of PRMT5 expression enhanced cell pyroptosis in MM. Moreover, we found that CASP1 expression was negatively correlated with PRMT5 expression, and repressing PRMT5 expression rescued both the phenotype and expression markers (N-GSDMD, IL-1b, and IL-18). Inhibition of PRMT5 activity increased CASP1 expression and promoted MM cell pyroptosis. Finally, high expression of PRMT5 or low expression of CASP1 was correlated with poor overall survival in MM. Collectively, our results provide a mechanism by which PRMT5 regulates cell pyroptosis by silencing CASP1 in MM.Subject terms: Myeloma, Enzyme mechanisms     

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.     

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.     

Versatility of PRMT5-induced methylation in growth control and development

Arginine methylation governs important cellular processes that impact growth and proliferation, as well as differentiation and development. Through their ability to catalyze symmetric or asymmetric methylation of histone and non-histone proteins, members of the protein arginine methyltransferase (PRMT) family regulate chromatin structure and expression of a wide spectrum of target genes. Unlike other PRMTs, PRMT5 works in concert with a variety of cellular proteins including ATP-dependent chromatin remodelers and co-repressors to induce epigenetic silencing. Recent work also implicates PRMT5 in the control of growth-promoting and pro-survival pathways, which demonstrates its versatility as an enzyme involved in both epigenetic regulation of anti-cancer target genes and organelle biogenesis. These studies not only provide insight into the molecular mechanisms by which PRMT5 contributes to growth control, but also justify therapeutic targeting of PRMT5.     

Theoretical Insights into Catalytic Mechanism of Protein Arginine Methyltransferase 1

Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical S_N2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.     

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.     

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.     

Discovery of new potent protein arginine methyltransferase 5 (PRMT5) inhibitors by assembly of key pharmacophores from known inhibitors

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been put into phase I clinical trial. In the present study, a series of candidate molecules were designed by combining key pharmacophores of formerly reported PRMT5 inhibitors. The in vitro PRMT5 inhibitory testing of compound 4b14 revealed an IC₅₀ of 2.71?μM, exhibiting high selectivity over PRMT1 and PRMT4 (>70-fold selective). As expected, 4b14 exhibited potent anti-proliferative activity against a panel of leukemia and lymphoma cells, including MV4-11, Pfeiffer, SU-DHL-4 and KARPAS-422. Besides, 4b14 showed significant cell cycle arrest and apoptosis-inducing effects, as well as reduced the cellular symmetric arginine dimethylation level of SmD3 protein. Finally, affinity profiling analysis indicated that hydrophobic interactions, π-π stacking and cation-π actions made the major contributions to the overall binding affinity. This scaffold provides a new chemical template for further development of better lead compounds targeting PRMT5.     

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.     

The Arginine Methyltransferase PRMT6 Cooperates with Polycomb Proteins in Regulating HOXA Gene Expression

Protein arginine methyltransferase 6 (PRMT6) catalyses asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a), which has been shown to impede the deposition of histone H3 lysine 4 trimethylation (H3K4me3) by blocking the binding and activity of the MLL1 complex. Importantly, the genomic occurrence of H3R2me2a has been found to coincide with histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone mark generated by the Polycomb repressive complex 2 (PRC2). Therefore, we investigate here a putative crosstalk between PRMT6- and PRC-mediated repression in a cellular model of neuronal differentiation. We show that PRMT6 and subunits of PRC2 as well as PRC1 are bound to the same regulatory regions of rostral HOXA genes and that they control the differentiation-associated activation of these genes. Furthermore, we find that PRMT6 interacts with subunits of PRC1 and PRC2 and that depletion of PRMT6 results in diminished PRC1/PRC2 and H3K27me3 occupancy and in increased H3K4me3 levels at these target genes. Taken together, our data uncover a novel, additional mechanism of how PRMT6 contributes to gene repression by cooperating with Polycomb proteins.