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.     

Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.     

A protein arginine N-methyltransferase 1 (PRMT1) and 2 heteromeric interaction increases PRMT1 enzymatic activity

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this study, we demonstrate a direct interaction between PRMT1 and PRMT2 using co-immunoprecipitation, bimolecular fluorescence complementation, and enzymatic assays. As a result of this interaction, PRMT2 stimulated PRMT1 activity, affecting its apparent V(max) and K(M) values in vitro and increasing the production of methylarginines in cells. Active site mutations and regional deletions from PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. Although the inhibition of methylation by adenosine dialdehyde prevented the interaction between PRMT1 and -2, it did not prevent the interaction between PRMT1 and a truncation mutant of PRMT2 lacking its Src homology 3 (SH3) domain. This result suggests that the SH3 domain may mediate an interaction between PRMT1 and -2 in a methylation-dependent fashion. On the basis of our findings, we propose that PRMT1 serves as the major methyltransferase in cells by forming higher-order oligomers with itself, PRMT2, and possibly other PRMTs.     

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H₂O₂ concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.     

Role for Btg1 and Btg2 in growth arrest of WEHI-231 cells through arginine methylation following membrane immunoglobulin engagement

Engagement of membrane Ig (mIg) on WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells, results in growth arrest and subsequent apoptosis. Of the several dozen genes upregulated greater than two-fold by anti-IgM treatment through DNA microarray analysis, we focused on B cell translocation gene 1 (Btg1) and Btg2, member of Btg/Tob family of proteins. WEHI-231 cells were infected with the Btg1/EGFP or Btg2/EGFP retroviral vectors, and those expressing either Btg1 or Btg2 accumulated in G1 phase at significantly higher proportions than that seen for cells expressing control vector. Btg1 or Btg2 bound to protein arginine methyltransferase (PRMT) 1 via the box C region, an interaction required for anti-IgM-induced growth inhibition. The arginine methyltransferase inhibitor AdOx partially abrogated growth inhibition induced by Btg1, Btg2, or anti-IgM. The Btg1- or Btg2-induced growth inhibition was also abrogated in PRMT1-deficient cells via introduction of small interference RNA. In addition, we observed anti-IgM-induced arginine methylation of two proteins, a 28-kDa and a 36-kDa protein. Methylation, detected by a monoclonal antibody specific for asymmetric, but not symmetric methyl residues, was observed as early as 1 h-2 h after stimulation and was sustained for up to 24 h. The anti-IgM-induced p36 arginine methylation was abrogated in the PRMT1-deficient cells, suggesting that PRMT1 induces p36 methylation. Together, these results suggest that anti-IgM-induced growth inhibition is mediated via upregulation of Btg1 and Btg2, resulting in the activation of arginine methyltransferase activity and culminating in growth inhibition of WEHI-231 cells.     

Arginine methylation of the cellular nucleic acid binding protein does not affect its subcellular localization but impedes RNA binding

Cellular nucleic acid binding protein (CNBP) contains seven zinc finger (ZF) repeats and an arginine and glycine (RG) rich sequence between the first and the second ZF. CNBP interacts with protein arginine methyltransferase PRMT1. Full-length but not RG-deleted or mutated CNBP can be methylated. Treatment with a methylation inhibitor AdOx reduced CNBP methylation, but did not affect the concentrated nuclear localization of CNBP. Nevertheless, arginine methylation of CNBP appeared to interfere with its RNA binding activity. Our findings show that arginine methylation of CNBP in the RG motif did not change the subcellular localization, but regulated its RNA binding activity.Structured summary of protein interactionsPRMT1 binds to CNBP by pull down (View interaction)PRMT1 methylates CNBP by enzymatic study (View interaction)CNBP physically interacts with PRMT1 by anti tag coimmunoprecipitation (View interaction)     

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.     

Design and synthesis of novel PRMT1 inhibitors and investigation of their binding preferences using molecular modelling

Protein arginine methyltransferase 1 (PRMT1) catalyses the methylation of substrate arginine by transferring the methyl group from SAM (S-adenosyl-l-methionine), which leads to the formation of S-adenosyl homocysteine (SAH) and methylated arginine. We have shown previously that the Asp84 on PRMT1 could be a potential inhibitor binding site. In the current study, 28 compounds were designed and synthesized that were predicted to bind the Asp84 and substrate arginine sites together. Among them, 6 compounds were identified as potential PRMT1 inhibitors, and showed strong inhibitory effects on cancer cell lines, especially HepG2. The most potent PRMT1 inhibitor, compound 13d, was selected for molecular dynamic simulations to investigate binding poses. Based on the free energy calculations and structural analysis, we predicted that the ethylenediamine group would tightly bind to Asp84, and the trifluoromethyl group should occupy part of substrate arginine binding site, which is consistent with our original goal. Our results show for the first time that PRMT1 inhibitors can target the Asp84 binding site, which will be helpful for future drug discovery studies.     

Protein arginine methylation regulates insulin signaling in L6 skeletal muscle cells

Protein N-arginine methyltransferase (PRMT)1 catalyzes arginine methylation in a variety of substrates, although the potential role of PRMT1 in insulin action has not been defined. We therefore investigated the effect of PRMT1-mediated methylation on insulin signaling and glucose uptake in skeletal L6 myotubes. Exposure of L6 myotubes to insulin rapidly induced translocation of PRMT1 and increased its catalytic activity in membrane fraction. Several proteins in the membrane fraction were arginine-methylated after insulin treatment, which were inhibited by pretreatment with an inhibitor of methyltransferase, 5′-deoxy-5′-(methylthio)adenosine (MTA), or a small interfering RNA against PRMT1 (PRMT1-siRNA). Inhibition of arginine methylation with MTA or PRMT1-siRNA diminished later phase of insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) β and IRS-1, association of IRS-1 with p85α subunit of PI3-K, and glucose uptake. Our results suggest that PRMT1-mediated methylation serves as a positive modulator of IR/IRS-1/PI3-K pathway and subsequent glucose uptake in skeletal muscle cells.     

The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization,Stress Granules,and Detergent-Insoluble Aggregates of FUS/TLS

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.     

Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells

Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced β-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.