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In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.

Members of the protein arginine methyltransferase (PRMT) family methylate the arginine residue(s) of several proteins and regulate a broad spectrum of cellular functions. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that asymmetrically dimethylates the arginine residues of numerous substrate proteins. PRMT6 introduces asymmetric dimethylation modification in the histone 3 at arginine 2 (H3R2me2a) and facilitates epigenetic regulation of global gene expression. In addition to histones, PRMT6 methylates a wide range of cellular proteins and regulates their functions. Here, we discuss (i) the biochemical aspects of enzyme kinetics, (ii) the structural features of PRMT6 and (iii) the diverse functional outcomes of PRMT6 mediated arginine methylation. Finally, we highlight how dysregulation of PRMT6 is implicated in various types of cancers and response to viral infections.

The eyes absent (EYA) family proteins are conserved transcriptional coactivators with intrinsic protein phosphatase activity. They play an essential role in the development of various organs in metazoans. These functions are associated with a unique combination of phosphatase and transactivation activities. However, it remains poorly understood how these activities and the consequent biologic functions of EYA are regulated. Here, we demonstrate that 2 conserved arginine residues, R304 and R306, of EYA1 are essential for its in vitro phosphatase activity and in vivo function during Drosophila eye development. EYA1 physically interacts with protein arginine methyltransferase 1, which methylates EYA1 at these residues both in vitro and in cultured mammalian and insect cells. Moreover, we show that wild‐type, but not methylation‐defective, EYA1 associates with γ‐H2A.X in response to ionizing radiation. Taken together, our results identify the conserved arginine residues of EYA1 that play an important role for its activity, thus implicating arginine methylation as a novel regulatory mechanism of EYA function.—Li, X., Eberhardt, A., Hansen, J. N., Bohmann, D., Li, H., Schor, N. F. Methylation of the phosphatase‐transcription activator EYA1 by protein arginine methyltransferase 1: mechanistic, functional, and structural studies. FASEB J. 31, 2327–2339 (2017). www.fasebj.org [ABSTRACT FROM AUTHOR]

Introduction: Arginine methylation is an important post-translational modification (PTM) in cells, which is catalyzed by a group of protein arginine methyltransferases (PRMTs). It plays significant roles in diverse cellular processes and various diseases. Misregulation and aberrant expression of PRMTs can provide potential biomarkers and therapeutic targets for drug discovery. Areas covered: Herein, we review the arginine methylation literature and summarize the methodologies for the characterization of this modification, as well as describe the recent insights into arginine methyltransferases and their biological functions in diseases. Expert commentary: Benefits from the enzyme-based large-scale screening approach, the novel affinity enrichment strategies, arginine methylated protein family is the focus of attention. Although a number of arginine methyltransferases and related substrates are identified, the catalytic mechanism of different types of PRMTs remains unclear and few related demethylases are characterized. Novel functional studies continuously reveal the importance of this modification in the cell cycle and diseases. A deeper understanding of arginine methylated proteins, modification sites, and their mechanisms of regulation is needed to explore their role in life processes, especially their relationship with diseases, thus accelerating the generation of potent, selective, cell-penetrant drug candidates. [ABSTRACT FROM PUBLISHER]

Protein arginine methylation is an important post-translational modification involved in different cellular processes, such as transcriptional regulation, RNA processing, DNA repair and signaling. Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs). However, little information on the involvement of PRMT-mediated protein arginine methylation in fruit ripening is available. Here, we identified and cloned nine PRMTs from the banana genome and characterized their phylogenetic relationship, conserved domains, gene structure and promoter cis-element. In addition, subcellular localization of MaPRMT proteins, expression profiles of MaPRMTs and protein arginine methylation levels during banana fruit ripening also were investigated. All the nine MaPRMTs were localized to cytoplasm and nucleus, suggesting that they might be involved in the histone and non-histone arginine methylation modification. The transcriptional levels of MaPRMT4B , MaPRMT6A and MaPRMT6B were significantly upregulated as fruit ripening proceeded. The upregulation was promoted by ethylene treatment, but postponed by 1-MCP treatment. Furthermore, the levels of protein arginine methylation, including asymmetric (aDMA) and symmetric (sDMA) dimethylarginine, were upregulated in banana during fruit ripening. These results suggested that PRMTs-mediated protein arginine methylation were implicated in banana fruit ripening. • Nine PRMT genes were isolated and confirmed from the banana genome. • All the 9 MaPRMTs were localized to cytoplasm and nuclear. • MaPRMT4B / 6A/6B were upregulated during fruit ripening. • aDMA and sDMA dimethylarginine levels were upregulated as fruit ripening proceeded. [ABSTRACT FROM AUTHOR]

Protein arginine methylation is a novel posttranslational modification that plays a pivotal role in a variety of intracellular events, such as signal transduction, protein-protein interaction and transcriptional regulation, either by the direct regulation of protein function or by metabolic products originating from protein arginine methylation that influence nitric oxide (NO)-dependent processes. A growing body of evidence suggests that both mechanisms are implicated in cardiovascular and pulmonary diseases. This review will present and discuss recent research on PRMTs and the methylation of non-histone proteins and its consequences for the pathogenesis of various lung disorders, including lung cancer, pulmonary fibrosis, pulmonary hypertension, chronic obstructive pulmonary disease and asthma. This article will also highlight novel directions for possible future investigations to evaluate the functional contribution of arginine methylation in lung homeostasis and disease. [ABSTRACT FROM AUTHOR]

Abstract: Protein arginine methylation is a major posttranslational modification that regulates various cellular functions, such as RNA processing and DNA repair. A recent report showed the involvement of protein arginine methyltransferase (PRMT) 4 in chromatin remodeling and gene expression during muscle differentiation in C2C12 cells. Because the fusion of myoblasts is a unique phenomenon observed in skeletal muscle differentiation, the present study focused on the expression and activities of PRMTs during myoblast fusion in primary rat skeletal muscle. NG, NG-asymmetric dimethylarginines (aDMA) and NG, N′G-symmetric dimethylarginines (sDMA) were both found consistently throughout myoblast fusion. However, PRMT1 exhibited the highest activity during myoblast fusion and maintained the elevated activity thereafter, whereas PRMT5 reached its highest activity only after myoblast fusion. To identify the proteins modified by such PRMTs, we conducted 2-dimensional electrophoresis (2-DE) of total proteins before and after myoblast fusion, and protein spots on the 2-DE gel immunoreactive for aDMA and sDMA were identified by mass spectrometric analysis. Among the proteins identified, lamin C2 was in particular observed to be dimethylated. Arginine methylation of lamin may therefore be important for muscle development and maintenance. [ABSTRACT FROM AUTHOR]

ABSTRACT The aim of the study was to identify as potential therapeutic targets specific molecular alterations in tumor cells recognized by the immune system. To identify such targets, we analyzed the human leukocyte antigen (HLA) peptidomes of human melanoma cells by 2-dimensional nano-HPLC/mass spectrometry and tested the immunological significance of the peptides by ex vivo ELISpot assays with lymphocytes from melanoma patients. The peptide SQNPRFYHK was identified as derived from the regulator of the nuclear corepressor complex (NCoR) G-protein pathway suppressor 2 (GPS-2) and to be differentially unmethylated, monomethylated or asymmetrically dimethylated at the arginine. The methylation state was specifically recognized by the immune system in that only the monomethylated variant induced T-cell responses and significantly stronger responses in patients than in healthy controls. The methylations were confirmed with synthetic analogues and in vitro radiolabeling assays with recombinant GPS-2 and synthetic peptides. The immunity of the 3 variants of GPS-2 was tested in T-cell assays with T lymphocytes of melanoma patients compared with healthy donors. The results show for the first time that GPS-2 is differentially methylated at a site that lacks known methylation motifs and that the methylation state is detected by the immune system. [ABSTRACT FROM AUTHOR]

Abstract: Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells. [Copyright &y& Elsevier]

The human 57 kDa Ki-1 antigen (Ki-1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki-1/57 interacts with chromo-helicase DNA-binding domain protein 3 and with the adaptor/signaling protein receptor of activated kinase 1 in the nucleus. Among the identified proteins that interacted with Ki-1/57 in a yeast two-hybrid system was the protein arginine-methyltransferase-1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two-hybrid system we were able to identify Ki-1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki-1/57 and its putative paralog CGI-55 have two conserved Gly/Arg-rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine-methylation by PRMT1. We observed that all Ki-1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki-1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine-2′,3′-dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki-1/57, whereas its paralog CGI-55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two-hybrid assay is an effective system for identifying novel PRMT arginine-methylation substrates and may be successfully applied to other members of the growing family of PRMTs. [ABSTRACT FROM AUTHOR]

Abstract: Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm. [Copyright &y& Elsevier]

Members of the protein arginine methyltransferase (PRMT) family methylate the arginine residue(s) of several proteins and regulate a broad spectrum of cellular functions. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that asymmetrically dimethylates the arginine residues of numerous substrate proteins. PRMT6 introduces asymmetric dimethylation modification in the histone 3 at arginine 2 (H3R2me2a) and facilitates epigenetic regulation of global gene expression. In addition to histones, PRMT6 methylates a wide range of cellular proteins and regulates their functions. Here, we discuss (i) the biochemical aspects of enzyme kinetics, (ii) the structural features of PRMT6 and (iii) the diverse functional outcomes of PRMT6 mediated arginine methylation. Finally, we highlight how dysregulation of PRMT6 is implicated in various types of cancers and response to viral infections. [ABSTRACT FROM AUTHOR]

PRMT7 is a member of the protein arginine methyltransferase (PRMT) family, which methylates a diverse set of substrates. Arginine methylation as a posttranslational modification regulates protein–protein and protein–nucleic acid interactions, and as such, has been implicated in various biological functions. PRMT7 is a unique, evolutionarily conserved PRMT family member that catalyzes the mono-methylation of arginine. The structural features, functional aspects, and compounds that inhibit PRMT7 are discussed here. Several studies have identified physiological substrates of PRMT7 and investigated the substrate methylation outcomes which link PRMT7 activity to the stress response and RNA biology. PRMT7-driven substrate methylation further leads to the biological outcomes of gene expression regulation, cell stemness, stress response, and cancer-associated phenotypes such as cell migration. Furthermore, organismal level phenotypes of PRMT7 deficiency have uncovered roles in muscle cell physiology, B cell biology, immunity, and brain function. This rapidly growing information on PRMT7 function indicates the critical nature of context-dependent functions of PRMT7 and necessitates further investigation of the PRMT7 interaction partners and factors that control PRMT7 expression and levels. Thus, PRMT7 is an important cellular regulator of arginine methylation in health and disease. [ABSTRACT FROM AUTHOR]

• An electrochemical assay is proposed for the detection of protein arginine methylation. • This assay, based on the Sakaguchi reaction, is highly selective towards the arginine methylation state. • This assay is simple, avoiding complex labelling or synthesis of reporting molecules. • This tool may facilitate the biological study of protein arginine methylases and demethylases. Protein arginine methylation is a common form of post-translational modification that plays an important role in many bioprocesses. However, research advances in this field have been severely hampered by the lack of a quick and sensitive method for detecting the arginine methylation state of a protein. In this work we propose a direct and sensitive electrochemical method for identifying the arginine methylation state. This novel assay combines an electrochemical technique with the Sakaguchi reaction, which is highly selective towards the arginine methylation state. We show that the presence of a methyl group on the arginine residue of a protein prevents the Sakaguchi reaction, while the unmethylated arginine residue selectively reacts with 8-hydroxyquinoline; the electrical signal of the reaction product is used for electrochemical detection. From this, a highly selective and simple electrochemical sensor has been developed based on (1) the high selectivity of the Sakaguchi reaction towards the arginine methylation state, and (2) the sensitive electrochemical signal generated by the linked 8-hydroxyquinoline. The assay described in this work thus provides a convenient tool for detection of protein arginine methylation, which may facilitate studies of the biological functions of protein arginine methylases and demethylases. [ABSTRACT FROM AUTHOR]