The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond

Cell. Mol. Life Sci. DOI 10.1007/s00018-015-1847-9 Cellular and Molecular Life Sciences REVIEW The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond Nicole Stopa • Jocelyn E. Krebs • David Shechter Received: 15 November 2014 / Revised: 10 January 2015 / Accepted: 29 January 2015  Springer Basel 2015 Abstract Post-translational arginine methylation is responsible for regulation of many biological processes. The protein arginine methyltransferase 5 (PRMT5, also known as Hsl7, Jbp1, Skb1, Capsuleen, or Dart5) is the major enzyme responsible for mono- and symmetric dimethylation of arginine. An expanding literature demonstrates its critical biological function in a wide range of cellular processes. Histone and other protein methylation by PRMT5 regulate genome organization, transcription, stem cells, primordial germ cells, differentiation, the cell cycle, and spliceosome assembly. Metazoan PRMT5 is found in complex with the WD-repeat protein MEP50 (also known as Wdr77, androgen receptor coactivator p44, or Valois). PRMT5 also directly associates with a range of other protein factors, including pICln, Menin, CoPR5 and RioK1 that may alter its subcellular localization and protein substrate selection. Protein substrate and PRMT5–MEP50 post-translation modifications induce crosstalk to regulate PRMT5 activity. Crystal structures of C. elegans PRMT5 and human and frog PRMT5–MEP50 complexes provide substantial insight into the mechanisms of substrate recognition and procession to dimethylation. Enzymological studies of PRMT5 have uncovered compelling insights essential for future development of specific N. Stopa  J. E. Krebs (&) Department of Biological Sciences, University of Alaska Anchorage, 3211 Providence Drive, Anchorage, AK 99508, USA e-mail: [email protected] D. Shechter (&) Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA e-mail: [email protected] PRMT5 inhibitors. In addition, newly accumulating evidence implicates PRMT5 and MEP50 expression levels and their methyltransferase activity in cancer tumorigenesis, and, significantly, as markers of poor clinical outcome, marking them as potential oncogenes. Here, we review the substantial new literature on PRMT5 and its partners to highlight the significance of understanding this essential enzyme in health and disease. Keywords Protein arginine methyltransferase  Histones  Spliceosome  Development  Cancer Introduction Protein arginine methyltransferases (PRMTs) transfer methyl groups from S-adenosylmethionine (AdoMet or SAM) to a guanidine nitrogen of protein arginine resulting in the reaction products methylarginine and S-adenosylhomocysteine (SAH) (reviewed in [1]). There are four types of PRMTs: type I PRMTs catalyze x-NGmonomethylarginine (MMA) and asymmetric x-NG, NGdimethylarginine (aDMA); type II PRMTs catalyze MMA and symmetric x-NG, N0 G-dimethylarginine (sDMA); type III PRMTs are capable of only monomethylation; and Type IV generates d-NG-monomethylarginine (Fig. 1; type IV activity, limited to yeast Rmt2 [2], is not shown). PRMT1, 2, 3, 4, 6, and 8 are Type I, while PRMT5 and possibly PRMT7 are Type II PRMTs [3–6]. Recent proteomic analysis of human tissues reveals differences in PRMT family protein expression (Fig. 2) [7], with higher expression in fetal tissues for all PRMTs. PRMT2, 3, 6, 7, and 8 exhibit tissue-specific expression patterns, while PRMT1, 4, and 5 exhibit more universal expression. PRMT5’s partner methylosome protein 50 (MEP50) has similar 123 N. Stopa et al. Fig. 1 Arginine methylation states catalyzed by the family of protein arginine methyltransferases (PRMTs). The guanidinium side chain of arginine residues in proteins is positively charged. It can accept a monomethyl addition, catalyzed by the family of Type I, II, and III PRMTs through transfer from the S-adenosylmethionine (SAM or AdoMet) cosubstrate, resulting in a x-NG monomethylated arginine (MMA) and S-adenosylhomocysteine (SAH). Type I PRMTs, comprising the majority of PRMT enzymes, can further catalyze the x-NG monomethylation to x-NG, NG asymmetric dimethylarginine (aDMA), consuming SAM and producing SAH. PRMT5, a Type II enzyme, catalyzes the x-NG monomethylation to x-NG, N0 G asymmetric dimethylarginine (sDMA), also consuming SAM and producing SAH. Type III enzymes are incapable of processing to dimethylation. Methylation does not alter the positive charge on the arginine guanidinium side chain Fig. 2 PRMT5 and MEP50 are broadly expressed in somatic and embryonic tissues. The human proteome map, analyzed by total proteome mass spectrometry (http://www.humanproteomemap.org [7]), was queried for the PRMT family of proteins which showed that they are distinctly expressed in a range of human tissues and cells. The relative protein abundances for the PRMT1-8 (CARM1 is the name for PRMT4) are shown in a heatmap, with white representing low protein abundance and dark red representing higher abundance, with a ten-step range indicated in the legend. PRMT5 is bolded and boxed, as is its MEP50 cofactor. Note that PRMT5 and MEP50 are most highly expressed in fetal tissue and that their expression patterns are quite similar expression to PRMT5. PRMT9, newly annotated in NCBI, is still undescribed. The initially annotated PRMT9 is now correctly identified as an F-box protein, FBXO11 [8]. PRMT5 is the primary Type II arginine methyltransferase and found in all eukaryotic species investigated (Fig. 3a). The S. cerevisiae homolog of PRMT5 is histone synthetic lethal 7 (Hsl7); the S. pombe homolog is Shk1 kinase-binding protein 1 (Skb1) [9, 10]. Hsl7’s synthetic lethality with histones that led to its name likely had no connection with histone methylation, as no evidence of 123 The PRMT5 arginine methyltransferase A B D C Fig. 3 PRMT5 domain organization and structure are evolutionarily conserved. a A range of PRMT5 protein sequences across eukaryotic species [Homo sapiens (human), Xenopus laevis (frog), Danio rerio (fish), Drosophila melanogaster (fly), Arabidopsis thaliana (plant), Caenorhabditis elegans (worm)] was aligned using the MAFFT algorithm and the Pam120 similarity index and represented in a heatmap from white (\60 % similarity) to dark blue (100 % similarity). Alignment gaps are indicated by a line, and overall identity is shown on the right. The major domains and interfaces are indicated above and below the sequences. Asterisk indicates sequence insertion in C. elegans PRMT5 that prohibits tetramerization. b The human prmt5 gene has multiple splice variants, as shown from the NCBI human genome sequence. All the variants are in the N-terminal domain of the encoded protein. c Subunit arrangement of the heterooctameric PRMT5–MEP50 structure shown in cartoon form, with the head-to-tail N-terminal and C-terminal PRMT5 arrangement shown by ‘‘N-’’ and ‘‘-C’’. d Ribbon diagram of a monomer of human PRMT5 (PDB:4GQB) with the domains and substrate-binding sites as indicated histone arginine methylation exists in S. cerevisiae. Human PRMT5 was first identified as Jak-binding protein 1 (JBP1), and shown to methylate, among many cellular proteins, histones H2A and H4 on Arg3 and histone H3 on Arg8 [11–13] (Table 1). Histones H2A and H4 share a conserved targeted N-terminal sequence: SGRGK…. Multiple PRMT5 splice variants are found in human cells, although evidence for translated proteins from these shorter mRNAs is lacking (Fig. 3b). In this review, we highlight and interpret the literature on PRMT5, its partners, targets, structure, and enzymology. We address PRMT5’s role in stem cells and primordial germ cells, differentiation, and animal development. In the context of PRMT5’s wide-ranging biological roles, we explore the extensive literature implicating PRMT5 in a large number of cancers. While hints of PRMT5’s significance for tumorigenesis have been apparent for some time, we argue here that the sheer abundance of evidence shows that PRMT5 is now a compelling target for clinical screening and, hopefully, for future chemotherapeutic approaches. A recent review of the function of all PRMTs in chromatin organization provides a complementary view of the specific function of arginine methylation in nuclear function [14]. MEP50: a critical PRMT5 cofactor The majority of vertebrate PRMT5 complexes contain MEP50, a 7-bladed WD40 repeat (tryptophan, aspartic acid) b-propeller protein. MEP50 is also known as Wdr77 or androgen receptor coactivator p44, by which it is referred to in the cancer literature [15–24]. MEP50 directly binds PRMT5 and greatly enhances PRMT5’s histone methyltransferase ability, primarily through increased 123 N. Stopa et al. Table 1 Major PRMT5 protein substrates and their function PRMT5 substrate Biological function of arginine methylation by PRMT5 References Histone H2A and H4 R3 Transcriptional repression [54, 56, 61, 62, 64, 65, 67, 68, 76, 77, 103, 162] Histone H3 R2 Transcriptional repression [4, 80, 163] Histone H3 R8 Transcriptional repression [13, 57, 60, 82, 122, 164] Spliceosome Sm proteins Facilitates spliceosomal assembly [41, 44, 49, 108, 165–169] Ribosomal protein RPS10 Facilitates ribosomal assembly [170] [71, 143] p53 Facilitates survival and cell cycle arrest over apoptosis FEN1 Facilitates PCNA binding and DNA replication and repair [171] Nucleoplasmin Enriched in early development; unknown function [37] Nucleolin RNA binding; unknown function [108, 109] EGFR Reduces autophosphorylation and EGFR activation [145] EBNA Methylation stimulates Epstein–Barr nuclear antigen promoted transcription [153, 154] affinity for protein substrate (D.S., manuscript under review). The arrangement of MEP50 in complex with PRMT5 is illustrated in Fig. 3c. Structure and enzymology of PRMT5 and MEP50 Structural insight into general PRMT mechanisms was recently reviewed [25]. The C. elegans, Xenopus, and human PRMT5 all contain a triosephosphate isomerase (TIM) barrel on the N-terminus, a middle Rossmann-fold, and a C-terminal b-barrel containing a dimerization domain (Fig. 3d). CePRMT5 forms a homodimer in which the dimerization arm of one monomer interacts with residues contained in the TIM barrel of the other monomer, forming a ring [26]. This head-to-tail ring-shaped homodimer is conserved in all of the solved Type I PRMT structures [27– 33]. In contrast, the human and Xenopus PRMT5s form a heterooctomeric complex composed of four PRMT5 proteins and four MEP50 proteins (Fig. 3c) [34, 35]. The PRMT5 molecules form two dimers in the head-to-tail arrangement typical of PRMTs. One of the two dimers in the human and Xenopus PRMT5 tetramer is similar to the C. elegans dimer and contains a number of conserved hydrogen bonds. The second dimer interface, unique to the human and Xenopus PRMT5 tetramer, contains hydrogen bonds not seen in the C. elegans dimer. Furthermore, a sequence insertion found in C. elegans would prevent this dimerization of PRMT5 to a tetramer (noted by asterisk in Fig. 3a). The PRMT5 tetramer forms the core of the complex and MEP50 interacts with PRMT5 through the N-terminal TIM barrel domains. A monomer of human PRMT5 is illustrated in Fig. 3d, showing the domain 123 structures as well as the locations of the SAM and histone peptide substrates within the crystal. The PRMT5–MEP50 complex has a higher level of methyltransferase activity compared to PRMT5 alone [35]. This could be due to MEP50 having a positive allosteric effect on the binding of cofactor and protein or SAM substrates by PRMT5 and/or MEP50 being necessary to present protein substrate to PRMT5. The latter is supported by experiments demonstrating MEP50 interaction with H2A and H4 [34, 36], and that excess MEP50 inhibits methyltransferase activity, consistent with MEP50 sequestering substrate from the enzyme [34]. The PRMT5 catalytic site is also oriented toward the cross-dimer paired MEP50 and electron microscopy-localized substrate density on MEP50 [34]. PRMT5–MEP50 is nonprocessive, as production of the dimethylated H4 peptide product is dependent on the concentration of the monomethylated peptide exceeding that of the unmethylated substrate [35, 37]. Thompson and colleagues [38] demonstrated that CePRMT5 is truly distributive. This is in contrast to PRMT1, for which monomethylated and dimethylated products are observed despite the presence of excess unmodified substrate, indicating PRMT1 uses a partially processive mechanism [39]. A conserved phenylalanine in the C. elegans PRMT5 catalytic site is essential for specifically catalyzing symmetric dimethylation by structural orientation of the monomethylated arginine substrate [26]. Mutation of a catalytic site Met to Phe remodels PRMT1 to produce symmetric dimethylation, although production of the symmetric dimethylarginine has a higher energy barrier [40]. This reveals that the catalytic mechanisms for production of the various methylarginine products are similar and are regulated through structural and energetic means. The PRMT5 arginine methyltransferase PRMT5 and the major spliceosome PRMT5–MEP50, along with PRMT7, play important roles in the splicing of mRNA through methylating spliceosomal proteins [41]. Sm proteins D1, D3, and B/B0 are symmetrically dimethylated on their C-terminus by the methylosome, PRMT5–MEP50 in complex with pICln (chloride channel nucleotide sensitive 1A, Fig. 4) [42, 43]. pICln binds the Sm domain and acts as an assembly chaperone [44–47]. PRMT5-catalyzed sDMA of Sm D1, D3, and B/B0 dramatically increases binding of these three proteins to the Tudor domain-containing protein SMN (survival of motor neuron), the product of the spinal muscular atrophy gene [42, 43]. SMN is part of a complex consisting of at least six other subunits, and is responsible for loading the seven Sm proteins onto the snRNA [48–51]. There is some evidence the snRNPs can assemble without the SMN complex in vitro [52], leading to some debate as to whether the symmetric dimethylation of Sm proteins is necessary. However, in vivo the SMN–PRMT5 relationship most likely acts as a chaperone that prevents the misassembly of Sm proteins to non-target RNAs and blocks the aggregation of Sm proteins [51]. A conditional PRMT5 knockout in mouse neural stem/progenitor cells (NPCs) shows PRMT5 is necessary for correct splicing: absence of PRMT5 leads to selective retention of introns and skipping of exons with weak 50 donor sites [53]. Histone methylation by PRMT5 and its function in transcriptional regulation Histone tail modifications are major components of the epigenetic regulation of gene transcription. PRMT5 symmetrically dimethylates H2AR3, H4R3, H3R2, and H3R8 in vivo, all of which are linked to a range of transcriptional regulatory events (Fig. 5) [11, 13, 54–60]. Specific gene targets include cyclin E1 [59], Rb [57], and ribosomal genes [61]. In Arabidopsis, PRMT5 is recruited to the CORYNE locus to down-regulate its expression and regulate shoot apical meristem phenotypes [62] and the FLOWERING LOCUS C to control flowering time [63]. PRMT5 coordinates with a range of Mediator complex subunits to dimethylate H4R3 at promoter regions of immune response genes and C/EBPb target genes [64]. Conversely, PRMT5 methylation of histone H3R2 recruits Wdr5 and the MLL complex, stimulating H3K4 methylation and euchromatin maintenance [4]. PRMT5 selectively methylates cytosolic H2AR3 in ES cells, but not H4R3 [65]. The distinction between roles for Fig. 4 PRMT5 methylation and regulation of the spliceosome. A cartoon representation of the function of PRMT5 methylation of splicing proteins in the cytoplasm. Methylated substrates are represented with a red ‘‘–CH3’’. PRMT5, in complex with MEP50 and pICln, form the methylosome that targets spliceosomal subunits for methylation. pICln then chaperones the subunits to the SMN complex, resulting in proper targeting of RNAs to be spliced 123 N. Stopa et al. Fig. 5 PRMT5 is targeted to multiple histone and nuclear targets by cofactors. A cartoon representation of the function of PRMT5 methylation of nuclear proteins (nucleus represented by pale yellow). Methylated substrates are represented with a red ‘‘–CH3’’. Histones, the protein component of chromatin, are synthesized and then transported to the nucleus. PRMT5–MEP50 targets newly synthesized histone H2A in the cytoplasm and may target soluble H4 in the nucleus (both H2A and H4 are methylated on R3 in the sequence N-SGRGK… as shown in the cartoon), as well as transcription factors such as p53 and NF-jB. PRMT5-methylated H2A and H4 are then deposited into chromatin (DNA wrapped around histone proteins, with histone N-terminal tails indicated in the cartoon). Alternative binding partners for PRMT5 (RioK1 in the cytoplasm, CoPR5 and Menin in the nucleus) may displace one or more MEP50 molecules and alter the targeting of PRMT5 toward substrates as shown, including histone H3 on R2 or R8 in the sequence N-ARTKQTARKST… H2A and H4 R3 methylation by PRMT5 suggests that each histone tail and targeted arginine has a unique function and will require future work to disentangle. However, since H2A and H4 have the same ‘‘NH2-SGRGK…’’ site of methylation, most available antibodies recognize both methylated histones making discrimination difficult. The few genome-wide studies of PRMT5-catalyzed histone methylation on H2A/H4 R3me2s demonstrate global enrichment [66], with specific enrichment at GC-rich promoter regions in mouse embryonic fibroblasts [67]. In contrast, enrichment on non-GC satellite DNA [68] as well as a modest anti-correlation with H3K36me3 [56] is observed in other studies. Girardot et al. [67] used an antibody lot that specifically recognizes H4R3me2s but not H2AR3me2s, possibly explaining these distinct observations. Future experimentation with a range of highly specific histone methylarginine antibodies, including monomethylarginine, and performed in a range of cell types and organisms, will help clarify the function of histone arginine methylation in gene regulation. PRMT5 also regulates transcription and many downstream events through methylation of transcription factors, such as NF-jB [69, 70], p53 [71], and E2F-1 [72]. PRMT1and PRMT5-catalyzed asymmetric and symmetric dimethylarginine have distinct roles in activating or suppressing apoptotic activity, respectively, of E2F-1 through recruitment of the p100-TSN Tudor domain to symmetric dimethylarginine [72]. 123 The PRMT5 arginine methyltransferase Readers of symmetric dimethylation Methylated arginine is translated into a meaningful cellular signal through recognition (‘‘reading’’) by effector proteins or by inhibiting binding of effector proteins (recently reviewed in [73]). Tudor domain-containing proteins are the primary direct readers of methylarginine. The splicing factors methylated by PRMT5 are recognized by SMN proteins containing Tudor domains [74] while PRMT5methylated PIWI proteins are recognized by the SND1 Tudor-containing protein [75]. Histone H4R3me2s specifically recruits the DNA methyltransferase DNMT3A to chromatin domains via its ADD (ATRX-DNMT3DNMT3L) domain to suppress gene expression [76, 77]. However, another report was unable to reproduce this interaction [78], so further study is necessary. In contrast, H4R3me2s or H4R3me2a can interfere with the ability of the Signal Recognition Particle (SRP) proteins SRP68 and SRP72 to bind the H4 tail [79]. PRMT5 also methylates histone H3R2 and recruits Wdr5, a WD40-repeat protein and essential component of MLL (mixed lineage leukemia lysine methyltransferase) complexes, to promote H3K4 methylation and downstream gene activation [4, 80]. Wdr5 quantitatively binds H3R2me2s, but does not bind H3R2me2a, providing a unique switch between recruitment states based on the change in methylarginine. The crystal structure of Wdr5 bound to H3R2me2s demonstrates that the symmetric dimethylarginine displaces water within the binding cavity, substantially enhancing the interaction and suggesting that WD-repeat proteins may function to distinguish between different post-translation modification (PTM) states [4]. Interaction of PRMT5 with ATP-dependent chromatin remodelers: function in transcriptional regulation PRMT5 methylates histones and interacts with ATP-dependent chromatin remodelers to either enable or repress gene expression, depending on the cellular context (Fig. 5) (reviewed in [81]). PRMT5 localizes to the promoter of the early MyoD-induced gene myogenin, and also coimmunoprecipitates with MyoD and the chromatin remodeler ATPase Brg1 [82]. Furthermore, H3R8 dimethylation catalyzed by PRMT5 at the myogenin promoter is a necessary prerequisite for the binding and chromatin remodeling activity of Brg1, which in turn is necessary for the binding of MyoD. Antisense-mediated knockdown of PRMT5 positively and negatively regulated many genes, including several with antiproliferative and tumor suppressor activity [13]; in this study, PRMT5 was shown to associate with the BRG1 and BRM chromatin remodelers and methylate promoter H3R8 to inhibit tumor suppressors. PRMT5 also associates with the NuRD remodeling complexes that contain the methyl-CpG-binding domain protein 2 (MBD2) [83]. Together these studies suggest that gene repression or activation by PRMT5 is context dependent. Other PRMTs associate with chromatin remodeling complexes as well. PRMT4 is required to facilitate SWI/ SNF chromatin remodeling activity for late but not early gene expression in skeletal muscle differentiation, in contrast to PRMT5 promotion of early gene expression [84, 85]. These data demonstrate that arginine methyltransferases sequentially cooperate with chromatin remodeling complexes. Role of PRMT5 in development PRMT5 participates in both early and late developmental pathways. In murine early development, PRMT5 is maternally inherited in the oocyte cytoplasm until the first cellular differentiation event when it translocates to the nucleus [65]. Prmt5-/- murine embryos suffer early embryonic lethality and are incapable of producing embryonic stem ES cells. RNAi knockdown of PRMT5 in ES cells results in down-regulation of pluripotency-associated genes and up-regulation of differentiation-associated genes [65]. In human stem cells, PRMT5 is only required for proliferation, and not pluripotency, through methylation of the cell cycle-regulated p57 [86]. Mep50 null mice are similarly embryonic lethal [21, 24], further supporting the essential function of the intact PRMT5–MEP50 complex. In Xenopus laevis embryos, prmt5 is abundant until zygotic stage 8, when transcript levels drop precipitously coincident with the onset of zygotic transcription [37]. PRMT5-methylated histones and histone chaperones are heavily enriched in early frog embryos [87–89]. PRMT5– MEP50 methylates pre-deposition histones H2A/H2A.X-F and H4 and the maternal histone chaperone nucleoplasmin on a conserved motif (‘‘GRGxK’’) [37]. These observations are consistent with a maternal and early zygotic role for PRMT5–MEP50 in regulating embryonic chromatin assembly and globally repressing zygotic transcription. PRMT5 function in primordial germ cell and keratinocyte differentiation PRMT5 also plays a role in a number of tissue-specific differentiation pathways, including primordial germ cells, keratinocyte, muscle, and nerve cell differentiation [81, 82, 84, 90–94]. In germ cell development, PRMT5 methylates Piwi proteins and regulates their subsequent binding to Tudor domain-containing proteins in an sDMA-dependent fashion 123 N. Stopa et al. [95–99]. Piwi proteins are primarily expressed in the germline lineage and interact with small non-coding RNAs, piRNAs [100]. piRNAs complement transposable DNA elements and other genes, leading to their silencing, which is essential for normal gametogenesis [101]. For example, in Drosophila, either a prmt5 homozygous null mutant or a loss of function Tudor mutation causes transposon upregulation [102]. PRMT5 histone methylation is also required for suppressing transposable elements during murine PGC demethylation [103]. PRMT5 interacts with the transcriptional repressor Blimp1, an essential component of primordial germ cell (PGC) induction [54, 104]. Association of PRMT5 and Blimp1 in the nucleus of PGCs results in increasing levels of H2A/H4 R3me2s and upon the subsequent translocation of PRMT5 and Blimp1 to the cytoplasm H2A/H4 R3me2s is almost completely lost [54]. This coincides with the down-regulation of pluripotency genes and the expression of Dhx38, an RNA helicase, which may recruit PRMT5 and Blimp1 to specific DNA sequences [54, 105]. These results suggest that the Blimp1/ PRMT5 complex has an essential role in maintaining the PGC lineage during the migration of the cells into the gonads [106]. Alternatively, PRMT5’s function may be at the end of PGC programming to regulate RNA splicing [107]. In human keratinocyte differentiation involucrin gene expression is partially controlled by PKC-d suppression of PRMT5 [92]. PRMT5 is part of the p38-d complex and functions through suppression of p38-d phosphorylation and sDMA modification of an as yet unidentified protein [92]. Modulation of PRMT5 activity through binding partners, post-translational modification crosstalk, and subcellular localization PRMT5 activity and localization are regulated in multiple ways, including binding partners (Table 2), PTMs, subcellular localization, and microRNAs (miRNA). Binding partner regulation of PRMT5 PRMT5 binds to pICln or the Rio domain-containing protein RioK1 in a mutually exclusive manner on PRMT5’s N-terminal domain, and likely serves to specify substrate choice [108] (Fig. 5). The RNA-binding protein nucleolin interacts only with the C-terminus of RioK1, and not with PRMT5 or MEP50. RioK1 functions similarly to pICln and MEP50 by acting as an adaptor protein [108]. In further support of the biological connection between PRMT5 and nucleolin, the AS1411 aptamer that targets nucleolin alters the subcellular localization of the PRMT5– 123 nucleolin complex within prostate cancer cells, potentially providing a molecular basis for some AS1411 effect on cancer cell proliferation [109]. RioK1 is exclusively located in the cytoplasm, which may further control the temporal and spatial activity of PRMT5. Therefore, coupled subcellular localization of adaptor proteins could be an important mechanism to regulate PRMT5 activity. Other vertebrate-specific binding partners also regulate or target PRMT5 activity to specific substrates, including Menin/Men1, pICln, RioK1, and CoPR5 [44, 45, 93, 108, 110–114]. CoPR5 (cooperator of PRMT5), to date only found in mammals, binds histones in the nucleus and recruits PRMT5 to nucleosomes [114]. CoPR5 binding to PRMT5 is necessary for myogenic differentiation, possibly through altered targeting of PRMT5 [93]. Menin, a unique adapter protein found in MLL complexes to target histone K4 trimethylation and frequently mutated in endocrine tumors, was shown to directly bind to the N-terminus of PRMT5 and target H4R3me2s at a specific promoter [110]. One compelling hypothesis supported by published interaction data and our structural modeling is that RioK1 and Menin may displace one or more MEP50 molecules from the PRMT5 complex, altering PRMT5 targeting while maintaining MEP50 in part of the heterocomplex to promote histone or other methylation (Fig. 5). This hypothesis could explain why PRMT5 forms a tetramer in vertebrates: to maintain MEP50 interaction and allow simultaneous binding of additional cofactors. Another mechanism for regulation of PRMT5 binding is via splicing. Alternative transcripts of PRMT5 missing exons in the N-terminus of PRMT5, which binds MEP50, Menin, Riok1 and plCln, are known (Fig. 3b) [115]. Future studies may reveal altered PRMT5 protein production from these transcripts that alter partner binding. PTM crosstalk modulation of PRMT5 PRMT5–MEP50 substrate PTMs can affect methyltransferase activity. SWI/SNF-associated PRMT5 methylates hypoacetylated H3 and H4 more efficiently than hyperacetylated H3 and H4 [58]. Neighboring H4 lysine acetylation marks stimulate PRMT5 activity in contrast to their inhibition of PRMT1 activity [116], while high-density histone peptide arrays document an elaborate crosstalk of activity regulation [34]. We modeled acetylation on H4K5 in the crystal structure of human PRMT5 and demonstrate that it would likely be stabilized in position compared to the hydrogen bonding with the structural water molecule in the unacetylated H4K5 in the structure (Fig. 6a, b). H2AS1 and H4S1 phosphorylation also inhibit PRMT5 activity [34]; as shown in Fig. 6c, the bulkier S1ph may be hindered from binding and/or may be electrostatically repulsed from the neighboring PRMT5 Y304. The PRMT5 arginine methyltransferase Table 2 Major PRMT5 interacting proteins and their function PRMT5 binding partnera Biological function References MEP50 (also known as Wdr77/Androgen Essential for PRMT5 histone methylation; Coactivator p44) always found bound to PRMT5 in metazoans [16, 22, 23, 34–37, 65, 83, 118, 125, 129, 167, 172, 173] pICln Contributes to spliceosome assembly and directs PRMT5 methylation to Sm proteins [44, 45, 47, 113, 167] RioK1 Competes with pICln for PRMT5 binding and recruits nucleolin for methylation [108] Menin/MEN1 Adapter protein for MLL methyltransferase that targets PRMT5 to chromatin [110–112] CoPR5 Mammalian nuclear protein that targets PRMT5 to chromatin [93, 174] hSWI/SNF Chromatin remodeling complexes Targets PRMT5 to chromatin and methylation of Histone H3 [13, 57, 58, 60, 82, 84, 122, 164] JAK kinases Mutant Jak2 found in leukemia phosphorylates PRMT5 and reduces its activity [11, 117] Blimp1 Localization of PRMT5 in primordial germ cells [54] AJUBA Piwi Coordinates PRMT5 interaction with SNAIL Recruitment via Tudor domain proteins to piRNA pathways [120] [95–97, 101] a Caution is warranted when considering PRMT5 interacting proteins identified in the literature by anti-FLAG precipitation (not shown here) as PRMT5 was shown to directly interact with FLAG antibodies [175] PTMs on PRMT5 or MEP50 also modulate methyltransferase activity. Although PRMT5 was first identified through its interaction with Jak2 protein in humans [11], the functional significance of this finding was not fully realized until recently. Mutant Jak2, common in certain types of leukemia, phosphorylates PRMT5 on its N-terminus in a region that is highly conserved from human to Xenopus (Y304 shown in Fig. 6c) [117]. This may abolish the interaction of PRMT5 with the histone substrate by clashing with its N-terminal Ser1 and thus significantly impairs the ability of PRMT5 to methylate histones H2A or H4 on R3 (similarly to H2A/H4 S1ph, Fig. 6c) [35, 117]. Conversely, phosphorylation of MEP50 on T5 increases the methyltransferase activity of PRMT5–MEP50 toward H4 [118], potentially by increased affinity for histone substrates. Finally, PRMT5 can influence the activity of other enzymes, as PRMT5 methylation of the transcription factor GATA4 inhibits p300-mediated GATA4 acetylation [119]. Subcellular localization and other regulation of PRMT5 In a variety of somatic cells, PRMT5 predominantly localizes to the cytoplasm [120–122] and as noted above the translocation of PRMT5 appears to play a role in controlling pluripotency in early development of mouse embryos [65]. PRMT5 has three novel nuclear exclusion signals (NES) that are unlike the conventional leucine-rich NES [123]. PRMT5 localization is also regulated by binding partners. The transcription factor SNAIL forms a complex with PRMT5–MEP50 mediated by the LIM protein AJUBA [120] and promotes translocation of the primarily cytoplasmic AJUBA and PRMT5 to the nucleus. SNAIL recruits the complex to the E-cadherin proximal promoter, resulting in increased methylation of H4R3. PRMT5 knockdown or inhibition results in expression of E-cadherin, suggesting transcriptional repression of E-cadherin by the SNAIL complex is dependent on PRMT5 methyltransferase activity. The SNAIL-induced epithelial-to-mesenchymal transition is essential during development and a major contributor to metastasis and tumor progression [124]. PRMT5 translation is regulated by miRNAs in mantle cell lymphoma (MCL) cells, in which a global increase in PRMT5 protein and H3R8 and H4R3 methylation appears despite less mRNA and slower transcription compared to normal B lymphocytes [122]. Re-expression of miRNAs that normally bind the 30 UTR of PRMT5 results in a strong decrease in PRMT5 protein levels. Similar results were obtained in transformed B cell chronic lymphocytic leukemia (B-CLL) cell lines [57]. Intriguingly, a prmt5 antisense RNA is found embedded within the prmt5 gene in the human genome possibly causing a similar effect on translation (NCBI Entrez Gene ID 100505758). PRMT5–MEP50 in cancer PRMT5’s regulation of proliferation and its direct interaction with proteins commonly misregulated or mutated in 123 N. Stopa et al. b Fig. 6 Structural basis for modification crosstalk regulation of A B C cancer indicate that PRMT5 may play a role in cancer as an oncogene [21–24, 57, 123, 125–129]. Cancer etiology is now highly correlated with alterations in the histone code signaling of epigenetic information [130, 131]. Yang and Bedford [132] provide an overall literature review of the role of the family of PRMTs in cancer. Increased expression and mutation of PRMT5 and MEP50 are found in a wide range of cancers, as we extracted from The Cancer Genome Atlas project database (Fig. 7a) [133]. PRMT5 overexpression appears to be an important factor in its tumorigenicity and occurs in a large 123 PRMT5 activity. The crystal structure of PRMT5–MEP50 complexed with H4 (1–8) tail peptide (PDB:4GQB) provided insight into activity crosstalk by other histone PTMs. a The histone H4 Lys 5 (H4K5, black stick) interacts with PRMT5 through a hydrogen bond between a structural water molecule (red ball) and its e-NH2. b Modeled interactions between an acetylated histone H4 Lys 5 (H4K5ac, yellow stick) within the HsPRMT5 active site. The oxygen-carbonyl occupies the position of the structural water molecule shown in a. Acetylation of the peptide at the K5 position increases the enzyme/substrate affinity through enhanced hydrogen bonding. c Modeled potential interactions between a phosphorylated histone H4 Ser 1 (H4S1ph) and the enzyme. The potential occupied space of the phosphorylated residue is shown in mesh, and may either sterically block histone peptide interaction, electrostatically repel PRMT5 Y304 in an active site pH-dependent fashion, or alternatively enhance interaction with enzyme and reduce turnover number of cancers, including ovarian, lung, lymphoid, lymphoma, glioblastoma multiforme, melanoma, colon, gastric, bladder cancer and germ cell tumors [57, 122, 123, 127–129, 134–138]. In epithelial ovarian cancer, elevated PRMT5 correlates with decreased patient survival [128]. Elevated PRMT5 and MEP50 expression in non-small cell lung cancer (NSCLC) is highly correlated (logrank P *2 9 10-6) with poorer survival in a large sample of patients, as we extracted from a clinical database of published data (http://www.kmplot.com, Fig. 7b, c) [139]. Mechanistic insight into this elevated expression in lung adenocarcinoma was shown by studies in which high cytoplasmic expression of PRMT5 was directly correlated with poor prognosis, possibly mediated through the epithelial-to mesenchymal transition [140] and histone methylation [141]. PRMT5 overexpression causes the formation of tumors in nude mice [135]. MEP50 had significant parallel roles in enhancing PRMT5 methylation of PI3-kinase to promote lung cancer tumorigenesis [142]. PRMT5 overexpression also results in increased proliferation and induced anchorage-independent colony growth [13, 135]. Conversely, PRMT5 knockdown significantly reduces cellular proliferation and colony formation in breast and lung cancer cells [13, 135, 143]. PRMT5 depletion inhibits proliferation in a majority of metastatic melanoma cell lines but accelerates growth in others [129]. These results suggest cell type might be an important factor in determining if overexpression leads to increased growth. However, no effect on cellular proliferation is observed when PRMT5 is overexpressed in MCF-7 breast cancer cells [143]. PRMT5 overexpression in cancer may in part be mediated by the NF-Y transcription factor, known to directly control cell cycle genes and other proliferative and cell survival factors [144]. PRMT5-catalyzed methylation of the growth factor receptor EGFR reduces its autophosphorylation, attenuating its activation and potentially playing a role in tumorigenesis [145]. The PRMT5 arginine methyltransferase A B C Fig. 7 PRMT5 is altered in a range of cancers and its expression is correlated with poor prognosis. a The alteration frequency of prmt5 gene amplification, mutation, and deletions in a wide range of human cancers cataloged in The Cancer Genome Atlas (TCGA, accessed through the cBio Cancer Genomics Portal; http://www.cbioportal.org) was plotted in a histogram, ranging up to 4.5 % alteration in uterine cancer. This analysis did not include increased gene expression or protein abundance. b A Kaplan–Meier survival probability plot for high (orange) versus low (gray) prmt5 gene expression/mRNA level for lung cancer is shown, with high prmt5 expression resulting in a *1.5-fold worse survival (hazard ratio) at very high significance. c A Kaplan–Meier survival probability plot for high (orange) versus low (gray) mep50 gene expression/mRNA level for lung cancer is shown, with high prmt5 expression resulting in a *1.6-fold worse survival (hazard ratio) at very high significance. Survival data obtained from http://www.kmplot.com The effect of PRMT5 overexpression on cellular proliferation suggests a role for PRMT5 in regulating cell cycle progression. PRMT5 knockdown slows the cell cycle in NIH3T3 cells and induces G1 arrest in 293T and MCF7 cells [135, 143]. PRMT5 overexpression increases the protein levels of the positive regulators of G1 phase cyclin D1, cyclin D2, cyclin E1, CDK4, and CDK6, and decreases the protein level of the negative regulator of G1 phase Rb protein [135]. Loss of PRMT5 leads to the increased expression of the cell cycle regulator p27Kip1 [129]. PRMT5 is also linked to the expression of the oncogenes p53, eukaryotic translation initiation factor (eIF4E), and microphthalmia-associated transcription factor (MITF) [129, 143, 146]. Knockdown of PRMT5 causes a significant decrease in both p53 and eIF4E [143]. Overexpression of eIF4E, a translational regulator, results in rapid proliferation, suppression of apoptosis, and malignant transformation [147, 148]. Expression of eIF4E rescues short-term loss of cellular proliferation caused by PRMT5 knockdown, consistent with eIF4E functioning as a critical downstream effector of PRMT5 activity [140]. In the human osteosarcoma cell line U2OS, PRMT5, Strap and p53 form a complex in response to DNA damage [71]. DNA damage-induced apoptosis is greater 123 N. Stopa et al. concomitant with PRMT5 knockdown, indicating that arginine methylation is a part of the p53 response. This apoptotic response could possibly be linked to PRMT5s role in splicing, such as in cell cycle genes with weak 50 donor sites. One of these mRNAs is Mdm4, which senses defects in the spliceosomal machinery and transfers the signal to activate the p53 response [53]. Furthermore, PRMT5 monomethylates p53 within its oligomerization domain on a similar ‘‘GRGR/K’’ sequence to that found in histones, modestly influencing p53 tetramer formation and its target selection [71]. PRMT5 activity is modulated by the DAL-1/4.1B tumor suppressor which is known to function in pro-apoptotic pathways in breast cancer cells [149, 150] and is essential for the growth of lung cancer cells [123, 135]. The programmed cell death 4 (PDCD4) tumor suppressor protein conversely functions to promote cell growth and tumor formation when overexpressed with PRMT5 [126, 151]. Menin/MEN1 interacts with PRMT5 to alter its activity, and cancer-associated Menin mutations appear to block this interaction, possibly altering the targeting of PRMT5 and promoting tumorigenesis [110, 111]. In developing fetal testes, both PRMT5 and MEP50 were nuclear in Leydig cells and in adult nonneoplastic testes; in contrast, testicular cancers exhibited reduced nuclear PRMT5 and MEP50 with enhanced cytoplasmic localization [125]. Similarly, cytoplasmic expression of MEP50 in prostate cancer cells promotes both androgenand estrogen-mediated transcriptional activity and tumorigenesis [17, 23], while forced nuclear localization of MEP50 inhibited prostate cancer cell proliferation [24]. Consistently, targeting PRMT5 to the nucleus by fusing a nuclear localization signal (NLS) to the N-terminus of PRMT5 also results in inhibition of growth of LNCaP cells. In contrast, MEP50 was nuclear in invasive ovarian and breast cancer cells while mainly cytoplasmic in normal cells [22]. Consistent with this observation, overexpression of MEP50 in the nucleus stimulated proliferation and invasion only in the presence of estrogen or androgen [19]. Part of the role of MEP50 in hormone-responsive tumors may be independent of PRMT5, mediated through interaction and recruitment of the Smad1 transcription factor [16]. PRMT5 in additional diseases and future drug design outlook Host and microbe PRMTs are involved in infectious disease pathways. Parasitic protozoa with PRMTs have a conserved Type I PRMT with homology to PRMT1 and a conserved Type II PRMT with homology to PRMT5 [152]. PRMT5 also binds and methylates the Epstein–Barr 123 Nuclear Antigen protein and stimulates EBNA-dependent transcription, possibly indicating that host PRMT5 plays a role in latent EB infection [153, 154]. Retroviral infections may also be regulated by PRMT5. Human T lymphotropic viruses encode accessory proteins p30 and p28, which were shown to interact specifically with PRMT5, while reduction of host cell PRMT5 levels decreased HTLV-2, but not HTLV-1, viral gene expression [155]. The HIV Tat protein is known to be methylated and regulated by PRMT6, and contains a long stretch of ‘‘GR’’ residues, suggesting that it may also be a target of PRMT5 [156]. PRMT5 may also have significance for heart disease. PRMT5, along with PRMT3, was shown to bind to and methylate the voltage-gated sodium channel NaV1.5. Strikingly, this arginine methylation enhanced NaV1.5 cell surface localization and current density, showing that this regulation may be a previously unknown component of heart health and disease [157]. PRMT5 also was shown to interact with GATA4 in cardiomyocytes and methylated it on three Arg residues, inhibiting the ability of GATA4 to promote transcriptional activation [119]. A number of other arginine and lysine methyltransferases have also been implicated in cancer and other diseases [132, 158, 159]. This makes PRMT5, and protein methyltransferases in general, a prime target for drug development and diagnostics [159]. Though no pharmacological treatments directly targeting PRMT5 are available yet, research into PRMT5 inhibitors has greatly increased within the last several years, with a number of inhibitors currently being developed specifically for application to cancer, b-thalassemia, or sickle cell disease. Interestingly, the epizyme inhibitor EPZ004777 directed against the Dot1L lysine methyltransferase also inhibits PRMT5, but not the PRMT5– MEP50 complex, suggesting that some of its activity may be due to PRMT5 inhibition [160, 161]. Concluding remarks Mono- and symmetric dimethylation of arginine is versatile and commonly utilized PTMs that until recently were under-recognized. An ever-greater number of proteins and cellular pathways are now known to be regulated by these modification states, including the splicing machinery and histones that are the foundation of many essential biological functions. Here, we focused on PRMT5 and highlighted its mechanisms of catalysis and substrate recognition, the somatic and cancerous biological processes that PRMT5 and its partner MEP50 participate in or are essential for, and showed the role PRMT5 and MEP50 play in early development. Current and forthcoming insights into PRMT5’s molecular mechanisms of targeting specific proteins and catalyzing mono- and dimethylation The PRMT5 arginine methyltransferase will provide crucial information for the development of specific small molecule inhibitors. Future research will clarify the role of PRMT5 in development and disease, while the development of specific small molecule inhibitors of PRMT5 may lead to novel chemotherapeutic approaches for cancer. However, caution is necessary in the potential use of specific PRMT5 inhibitors due to their multiple biological roles, suggesting possible toxicity from its inhibition. New studies targeting PRMT5, and redundancy with other methyltransferases such as PRMT7, and their multiple biological roles are necessary to fully understand how PRMT5 functions in health and disease. New tools, such as better methylarginine antibodies that can distinguish histone substrates and mono- and dimethylation states, as well as conditional knockouts in cell culture and animals will be essential for future elucidation of the important biological roles of PRMT5. Acknowledgments N.S. and J.K. were supported by NIH/NIGMS [P20GM103395]. D.S. is funded by an NIH/NIGMS grant [R01GM108646] and by The American Cancer Society—Robbie Sue Mudd Kidney Cancer Research Scholar Grant [124891-RSG-13-39601-DMC]. We are grateful to Emmanuel Burgos for structural and enzymatic insight and for rendering Fig. 6. The analysis shown in Fig. 7 is based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov and from KM-plotter: http://kmplot. com. We thank the specimen donors to these projects for their essential contributions. We thank the many investigators studying PRMT5 and we apologize to the authors whose work on PRMT5 was not included due to space limitations. References 1. Di Lorenzo A, Bedford MT (2011) Histone arginine methylation. FEBS Lett 585(13):2024–2031. doi:10.1016/j.febslet.2010. 11.010 2. Niewmierzycka A, Clarke S (1999) S-Adenosylmethionine-dependent methylation in Saccharomyces cerevisiae. Identification of a novel protein arginine methyltransferase. J Biol Chem 274(2):814–824 3. Bedford MT, Clarke SG (2009) Protein arginine methylation in mammals: who, what, and why. Mol Cell 33(1):1–13. doi:10. 1016/j.molcel.2008.12.013 4. Migliori V, Muller J, Phalke S, Low D, Bezzi M, Mok WC, Sahu SK, Gunaratne J, Capasso P, Bassi C, Cecatiello V, De Marco A, Blackstock W, Kuznetsov V, Amati B, Mapelli M, Guccione E (2012) Symmetric dimethylation of H3R2 is a newly identified histone mark that supports euchromatin maintenance. Nat Struct Mol Biol 19(2):136–144. doi:10.1038/nsmb. 2209 5. Zurita-Lopez CI, Sandberg T, Kelly R, Clarke SG (2012) Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming omega-NG-monomethylated arginine residues. J Biol Chem 287(11):7859–7870. doi:10.1074/jbc.M111.336271 6. Feng Y, Maity R, Whitelegge JP, Hadjikyriacou A, Li Z, ZuritaLopez C, Al-Hadid Q, Clark AT, Bedford MT, Masson JY, Clarke SG (2013) Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions. J Biol Chem 288(52):37010–37025. doi:10.1074/jbc.M113.525345 7. Kim MS, Pinto SM, Getnet D, Nirujogi RS, Manda SS, Chaerkady R, Madugundu AK, Kelkar DS, Isserlin R, Jain S, Thomas JK, Muthusamy B, Leal-Rojas P, Kumar P, Sahasrabuddhe NA, Balakrishnan L, Advani J, George B, Renuse S, Selvan LD, Patil AH, Nanjappa V, Radhakrishnan A, Prasad S, Subbannayya T, Raju R, Kumar M, Sreenivasamurthy SK, Marimuthu A, Sathe GJ, Chavan S, Datta KK, Subbannayya Y, Sahu A, Yelamanchi SD, Jayaram S, Rajagopalan P, Sharma J, Murthy KR, Syed N, Goel R, Khan AA, Ahmad S, Dey G, Mudgal K, Chatterjee A, Huang TC, Zhong J, Wu X, Shaw PG, Freed D, Zahari MS, Mukherjee KK, Shankar S, Mahadevan A, Lam H, Mitchell CJ, Shankar SK, Satishchandra P, Schroeder JT, Sirdeshmukh R, Maitra A, Leach SD, Drake CG, Halushka MK, Prasad TS, Hruban RH, Kerr CL, Bader GD, IacobuzioDonahue CA, Gowda H, Pandey A (2014) A draft map of the human proteome. Nature 509(7502):575–581. doi:10.1038/ nature13302 8. Cook JR, Lee JH, Yang ZH, Krause CD, Herth N, Hoffmann R, Pestka S (2006) FBXO11/PRMT9, a new protein arginine methyltransferase, symmetrically dimethylates arginine residues. Biochem Biophys Res Commun 342(2):472–481. doi:10. 1016/j.bbrc.2006.01.167 9. Ma XJ, Lu Q, Grunstein M (1996) A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae. Genes Dev 10(11):1327–1340 10. Gilbreth M, Yang P, Bartholomeusz G, Pimental RA, Kansra S, Gadiraju R, Marcus S (1998) Negative regulation of mitosis in fission yeast by the shk1 interacting protein skb1 and its human homolog, Skb1Hs. Proc Natl Acad Sci USA 95(25):14781–14786 11. Pollack BP, Kotenko SV, He W, Izotova LS, Barnoski BL, Pestka S (1999) The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity. J Biol Chem 274(44):31531–31542 12. Branscombe TL, Frankel A, Lee JH, Cook JR, Yang Z, Pestka S, Clarke S (2001) PRMT5 (Janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins. J Biol Chem 276(35):32971–32976. doi:10.1074/jbc. M105412200 13. Pal S, Vishwanath SN, Erdjument-Bromage H, Tempst P, Sif S (2004) Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes. Mol Cell Biol 24(21):9630–9645. doi:10.1128/mcb.24.21.9630-9645.2004 14. Jahan S, Davie JR (2014) Protein arginine methyltransferases (PRMTs): role in chromatin organization. Adv Biol Regul. doi:10.1016/j.jbior.2014.09.003 15. Zhou L, Hosohata K, Gao S, Gu Z, Wang Z (2013) cGMPdependent protein kinase Ibeta interacts with p44/WDR77 to regulate androgen receptor-driven gene expression. PLoS ONE 8(6):e63119. doi:10.1371/journal.pone.0063119 16. Li Y, Tian L, Ligr M, Daniels G, Peng Y, Wu X, Singh M, Wei J, Shao Y, Lepor H, Xu R, Chang Z, Wang Z, Lee P (2013) Functional domains of androgen receptor coactivator p44/ Mep50/WDR77 and its interaction with Smad1. PLoS ONE 8(5):e64663. doi:10.1371/journal.pone.0064663 17. Gao S, Wang Z (2012) Subcellular localization of p44/WDR77 determines proliferation and differentiation of prostate epithelial cells. PLoS ONE 7(11):e49173. doi:10.1371/journal.pone. 0049173 18. Vincent B, Wu H, Gao S, Wang Z (2012) Loss of the androgen receptor cofactor p44/WDR77 induces astrogliosis. Mol Cell Biol 32(17):3500–3512. doi:10.1128/mcb.00298-12 19. Ligr M, Patwa RR, Daniels G, Pan L, Wu X, Li Y, Tian L, Wang Z, Xu R, Wu J, Chen F, Liu J, Wei JJ, Lee P (2011) 123 N. Stopa et al. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. Expression and function of androgen receptor coactivator p44/ Mep50/WDR77 in ovarian cancer. PLoS ONE 6(10):e26250. doi:10.1371/journal.pone.0026250 Gu Z, Zhou L, Gao S, Wang Z (2011) Nuclear transport signals control cellular localization and function of androgen receptor cofactor p44/WDR77. PLoS ONE 6(7):e22395. doi:10.1371/ journal.pone.0022395 Gao S, Wu H, Wang F, Wang Z (2010) Altered differentiation and proliferation of prostate epithelium in mice lacking the androgen receptor cofactor p44/WDR77. Endocrinology 151(8):3941–3953. doi:10.1210/en.2009-1080 Peng Y, Li Y, Gellert LL, Zou X, Wang J, Singh B, Xu R, Chiriboga L, Daniels G, Pan R, Zhang DY, Garabedian MJ, Schneider RJ, Wang Z, Lee P (2010) Androgen receptor coactivator p44/Mep50 in breast cancer growth and invasion. J Cell Mol Med 14(12):2780–2789. doi:10.1111/j.1582-4934.2009. 00936.x Peng Y, Chen F, Melamed J, Chiriboga L, Wei J, Kong X, McLeod M, Li Y, Li CX, Feng A, Garabedian MJ, Wang Z, Roeder RG, Lee P (2008) Distinct nuclear and cytoplasmic functions of androgen receptor cofactor p44 and association with androgen-independent prostate cancer. Proc Natl Acad Sci USA 105(13):5236–5241. doi:10.1073/pnas.0712262105 Zhou L, Wu H, Lee P, Wang Z (2006) Roles of the androgen receptor cofactor p44 in the growth of prostate epithelial cells. J Mol Endocrinol 37(2):283–300. doi:10.1677/jme.1.02062 Schapira M, Ferreira de Freitas R (2014) Structural biology and chemistry of protein arginine methyltransferases. MedChemComm. doi:10.1039/C4MD00269E Sun L, Wang M, Lv Z, Yang N, Liu Y, Bao S, Gong W, Xu RM (2011) Structural insights into protein arginine symmetric dimethylation by PRMT5. Proc Natl Acad Sci USA 108(51):20538–20543. doi:10.1073/pnas.1106946108 Zhang X, Cheng X (2003) Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides. Structure 11(5):509–520 pii: S0969212603000716 Zhang X, Zhou L, Cheng X (2000) Crystal structure of the conserved core of protein arginine methyltransferase PRMT3. EMBO J 19(14):3509–3519. doi:10.1093/emboj/19.14.3509 Yue WW, Hassler M, Roe SM, Thompson-Vale V, Pearl LH (2007) Insights into histone code syntax from structural and biochemical studies of CARM1 methyltransferase. EMBO J 26(20):4402–4412. doi:10.1038/sj.emboj.7601856 Troffer-Charlier N, Cura V, Hassenboehler P, Moras D, Cavarelli J (2007) Functional insights from structures of coactivator-associated arginine methyltransferase 1 domains. EMBO J 26(20):4391–4401. doi:10.1038/sj.emboj.7601855 Cheng Y, Frazier M, Lu F, Cao X, Redinbo MR (2011) Crystal structure of the plant epigenetic protein arginine methyltransferase 10. J Mol Biol 414(1):106–122. doi:10.1016/j.jmb.2011. 09.040 Siarheyeva A, Senisterra G, Allali-Hassani A, Dong A, Dobrovetsky E, Wasney GA, Chau I, Marcellus R, Hajian T, Liu F, Korboukh I, Smil D, Bolshan Y, Min J, Wu H, Zeng H, Loppnau P, Poda G, Griffin C, Aman A, Brown PJ, Jin J, Al-Awar R, Arrowsmith CH, Schapira M, Vedadi M (2012) An allosteric inhibitor of protein arginine methyltransferase 3. Structure 20(8):1425–1435. doi:10.1016/j.str.2012.06.001 Wang C, Zhu Y, Chen J, Li X, Peng J, Chen J, Zou Y, Zhang Z, Jin H, Yang P, Wu J, Niu L, Gong Q, Teng M, Shi Y (2014) Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei. PLoS ONE 9(2):e87267. doi:10.1371/journal. pone.0087267 Ho M-C, Wilczek C, Bonanno JB, Xing L, Seznec J, Matsui T, Carter LG, Onikubo T, Kumar PR, Chan MK, Brenowitz M, 123 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. Cheng RH, Reimer U, Almo SC, Shechter D (2013) Structure of the arginine methyltransferase PRMT5–MEP50 reveals a mechanism for substrate specificity. PLoS ONE 8(2):e57008. doi:10.1371/journal.pone.0057008 Antonysamy S, Bonday Z, Campbell RM, Doyle B, Druzina Z, Gheyi T, Han B, Jungheim LN, Qian Y, Rauch C, Russell M, Sauder JM, Wasserman SR, Weichert K, Willard FS, Zhang A, Emtage S (2012) Crystal structure of the human PRMT5:MEP50 complex. Proc Natl Acad Sci USA 109(44):17960–17965. doi:10.1073/pnas.1209814109 Furuno K, Masatsugu T, Sonoda M, Sasazuki T, Yamamoto K (2006) Association of Polycomb group SUZ12 with WD-repeat protein MEP50 that binds to histone H2A selectively in vitro. Biochem Biophys Res Commun 345(3):1051–1058. doi:10. 1016/j.bbrc.2006.05.014 Wilczek C, Chitta R, Woo E, Shabanowitz J, Chait BT, Hunt DF, Shechter D (2011) Protein arginine methyltransferase Prmt5–Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs. J Biol Chem 286(49):42221–42231. doi:10.1074/jbc.M111.303677 Wang M, Xu RM, Thompson PR (2013) Substrate specificity, processivity, and kinetic mechanism of protein arginine methyltransferase 5. Biochemistry 52(32):5430–5440. doi:10. 1021/bi4005123 Osborne TC, Obianyo O, Zhang X, Cheng X, Thompson PR (2007) Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis. Biochemistry 46(46):13370–13381. doi:10.1021/bi701558t Gui S, Gathiaka S, Li J, Qu J, Acevedo O, Hevel JM (2014) A remodeled protein arginine methyltransferase 1 (PRMT1) generates symmetric dimethylarginine. J Biol Chem 289(13):9320–9327. doi:10.1074/jbc.M113.535278 Gonsalvez GB, Tian L, Ospina JK, Boisvert FM, Lamond AI, Matera AG (2007) Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins. J Cell Biol 178(5):733–740. doi:10.1083/jcb.200702147 Brahms H, Meheus L, de Brabandere V, Fischer U, Luhrmann R (2001) Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B0 and the Sm-like protein LSm4, and their interaction with the SMN protein. RNA 7(11):1531–1542 Friesen WJ, Massenet S, Paushkin S, Wyce A, Dreyfuss G (2001) SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets. Mol Cell 7(5):1111–1117. doi:10.1016/S10972765(01)00244-1 Meister G, Eggert C, Buhler D, Brahms H, Kambach C, Fischer U (2001) Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln. Curr Biol 11(24):1990–1994 Friesen WJ, Paushkin S, Wyce A, Massenet S, Pesiridis GS, Van Duyne G, Rappsilber J, Mann M, Dreyfuss G (2001) The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins. Mol Cell Biol 21(24):8289–8300. doi:10.1128/mcb.21.24.8289-8300.2001 Chari A, Golas MM, Klingenhager M, Neuenkirchen N, Sander B, Englbrecht C, Sickmann A, Stark H, Fischer U (2008) An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs. Cell 135(3):497–509. doi:10. 1016/j.cell.2008.09.020 Grimm C, Chari A, Pelz JP, Kuper J, Kisker C, Diederichs K, Stark H, Schindelin H, Fischer U (2013) Structural basis of assembly chaperone-mediated snRNP formation. Mol Cell 49(4):692–703. doi:10.1016/j.molcel.2012.12.009 Meister G, Buhler D, Pillai R, Lottspeich F, Fischer U (2001) A multiprotein complex mediates the ATP-dependent assembly of The PRMT5 arginine methyltransferase 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. spliceosomal U snRNPs. Nat Cell Biol 3(11):945–949. doi:10. 1038/ncb1101-945 Meister G, Fischer U (2002) Assisted RNP assembly: SMN and PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs. EMBO J 21(21):5853–5863 Gubitz AK, Feng W, Dreyfuss G (2004) The SMN complex. Exp Cell Res 296(1):51–56. doi:10.1016/j.yexcr.2004.03.022 Pellizzoni L, Yong J, Dreyfuss G (2002) Essential role for the SMN complex in the specificity of snRNP assembly. Science 298(5599):1775–1779. doi:10.1126/science.1074962 Raker VA, Plessel G, Luhrmann R (1996) The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J 15(9):2256–2269 Bezzi M, Teo SX, Muller J, Mok WC, Sahu SK, Vardy LA, Bonday ZQ, Guccione E (2013) Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 premRNA in sensing defects in the spliceosomal machinery. Genes Dev 27(17):1903–1916. doi:10.1101/gad.219899.113 Ancelin K, Lange UC, Hajkova P, Schneider R, Bannister AJ, Kouzarides T, Surani MA (2006) Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat Cell Biol 8(6):623–630. doi:10.1038/ncb1413 Bedford MT (2007) Arginine methylation at a glance. J Cell Sci 120(Pt 24):4243–4246. doi:10.1242/jcs.019885 Xu X, Hoang S, Mayo MW, Bekiranov S (2010) Application of machine learning methods to histone methylation ChIP-Seq data reveals H4R3me2 globally represses gene expression. BMC Bioinform 11:396. doi:10.1186/1471-2105-11-396 Wang L, Pal S, Sif S (2008) Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells. Mol Cell Biol 28(20):6262–6277. doi:10.1128/mcb.00923-08 Pal S, Yun R, Datta A, Lacomis L, Erdjument-Bromage H, Kumar J, Tempst P, Sif S (2003) mSin3A/histone deacetylase 2- and PRMT5containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad. Mol Cell Biol 23(21):7475–7487 Fabbrizio E, El Messaoudi S, Polanowska J, Paul C, Cook JR, Lee JH, Negre V, Rousset M, Pestka S, Le Cam A, Sardet C (2002) Negative regulation of transcription by the type II arginine methyltransferase PRMT5. EMBO Rep 3(7):641–645. doi:10.1093/embo-reports/kvf136 Seth-Vollenweider T, Joshi S, Dhawan P, Sif S, Christakos S (2014) Novel mechanism of negative regulation of 1,25-dihydroxyvitamin D3 induced CYP24A1 transcription: epigenetic modification involving crosstalk between protein arginine methyltransferase 5 and the SWI/SNF complex. J Biol Chem. doi:10.1074/jbc.M114.583302 Majumder S, Alinari L, Roy S, Miller T, Datta J, Sif S, Baiocchi R, Jacob ST (2010) Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription. J Cell Biochem 109(3):553–563. doi:10.1002/jcb.22432 Yue M, Li Q, Zhang Y, Zhao Y, Zhang Z, Bao S (2013) Histone H4R3 methylation catalyzed by SKB1/PRMT5 is required for maintaining shoot apical meristem. PLoS ONE 8(12):e83258. doi:10.1371/journal.pone.0083258 Wang X, Zhang Y, Ma Q, Zhang Z, Xue Y, Bao S, Chong K (2007) SKB1-mediated symmetric dimethylation of histone H4R3 controls flowering time in Arabidopsis. EMBO J 26(7):1934–1941. doi:10.1038/sj.emboj.7601647 Tsutsui T, Fukasawa R, Shinmyouzu K, Nakagawa R, Tobe K, Tanaka A, Ohkuma Y (2013) Mediator complex recruits epigenetic regulators via its two cyclin-dependent kinase subunits to repress transcription of immune response genes. J Biol Chem 288(29):20955–20965. doi:10.1074/jbc.M113.486746 65. Tee WW, Pardo M, Theunissen TW, Yu L, Choudhary JS, Hajkova P, Surani MA (2010) Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev 24(24):2772–2777. doi:10.1101/gad. 606110 66. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K (2007) High-resolution profiling of histone methylations in the human genome. Cell 129(4):823–837 67. Girardot M, Hirasawa R, Kacem S, Fritsch L, Pontis J, Kota SK, Filipponi D, Fabbrizio E, Sardet C, Lohmann F, Kadam S, AitSi-Ali S, Feil R (2014) PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G ? C-rich regions of the mouse genome. Nucleic Acids Res 42(1):235–248. doi:10.1093/nar/gkt884 68. Capurso D, Xiong H, Segal MR (2012) A histone arginine methylation localizes to nucleosomes in satellite II and III DNA sequences in the human genome. BMC Genom 13:630. doi:10. 1186/1471-2164-13-630 69. Harris DP, Bandyopadhyay S, Maxwell TJ, Willard B, DiCorleto PE (2014) Tumor necrosis factor (TNF)-alpha induction of CXCL10 in endothelial cells requires protein arginine methyltransferase 5 (PRMT5)-mediated nuclear factor (NF)-kappaB p65 methylation. J Biol Chem 289(22):15328–15339. doi:10. 1074/jbc.M114.547349 70. Wei H, Wang B, Miyagi M, She Y, Gopalan B, Huang DB, Ghosh G, Stark GR, Lu T (2013) PRMT5 dimethylates R30 of the p65 subunit to activate NF-kappaB. Proc Natl Acad Sci USA 110(33):13516–13521. doi:10.1073/pnas.1311784110 71. Jansson M, Durant ST, Cho EC, Sheahan S, Edelmann M, Kessler B, La Thangue NB (2008) Arginine methylation regulates the p53 response. Nat Cell Biol 10(12):1431–1439. doi:10.1038/ncb1802 72. Zheng S, Moehlenbrink J, Lu YC, Zalmas LP, Sagum CA, Carr S, McGouran JF, Alexander L, Fedorov O, Munro S, Kessler B, Bedford MT, Yu Q, La Thangue NB (2013) Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1. Mol Cell 52(1):37–51. doi:10.1016/j.molcel.2013.08. 039 73. Gayatri S, Bedford MT (2014) Readers of histone methylarginine marks. Biochim Biophys Acta 1839(8):702–710. doi:10.1016/j.bbagrm.2014.02.015 74. Côté J, Richard S (2005) Tudor domains bind symmetrical dimethylated arginines. J Biol Chem 280(31):28476–28483. doi:10.1074/jbc.M414328200 75. Liu K, Chen C, Guo Y, Lam R, Bian C, Xu C, Zhao DY, Jin J, MacKenzie F, Pawson T, Min J (2010) Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain. Proc Natl Acad Sci USA 107(43):18398–18403. doi:10.1073/pnas.1013106107 76. Zhao Q, Rank G, Tan YT, Li H, Moritz RL, Simpson RJ, Cerruti L, Curtis DJ, Patel DJ, Allis CD, Cunningham JM, Jane SM (2009) PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing. Nat Struct Mol Biol 16(3):304–311. doi:10.1038/nsmb. 1568 77. Rank G, Cerruti L, Simpson RJ, Moritz RL, Jane SM, Zhao Q (2010) Identification of a PRMT5-dependent repressor complex linked to silencing of human fetal globin gene expression. Blood 116(9):1585–1592. doi:10.1182/blood-2009-10-251116 78. Otani J, Nankumo T, Arita K, Inamoto S, Ariyoshi M, Shirakawa M (2009) Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRXDNMT3-DNMT3L domain. EMBO Rep 10(11):1235–1241. doi:10.1038/embor.2009.218 123 N. Stopa et al. 79. Li J, Zhou F, Zhan D, Gao Q, Cui N, Li J, Iakhiaeva E, Zwieb C, Lin B, Wong J (2012) A novel histone H4 arginine 3 methylation-sensitive histone H4 binding activity and transcriptional regulatory function for signal recognition particle subunits SRP68 and SRP72. J Biol Chem 287(48):40641–40651. doi:10. 1074/jbc.M112.414284 80. Yuan C-C, Matthews Adam GW, Jin Y, Chen Chang F, Chapman Brad A, Ohsumi Toshiro K, Glass Karen C, Kutateladze Tatiana G, Borowsky Mark L, Struhl K, Oettinger Marjorie A (2012) Histone H3R2 symmetric dimethylation and histone H3K4 trimethylation are tightly correlated in eukaryotic genomes. Cell Reports 1(2):83–90 81. Karkhanis V, Hu Y-J, Baiocchi RA, Imbalzano AN, Sif S (2011) Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem Sci 36(12):633–641. doi:10. 1016/j.tibs.2011.09.001 82. Dacwag CS, Ohkawa Y, Pal S, Sif S, Imbalzano AN (2007) The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling. Mol Cell Biol 27(1):384–394. doi:10.1128/mcb. 01528-06 83. Le Guezennec X, Vermeulen M, Brinkman AB, Hoeijmakers WA, Cohen A, Lasonder E, Stunnenberg HG (2006) MBD2/ NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties. Mol Cell Biol 26(3):843–851. doi:10.1128/mcb.26.3.843-851.2006 84. Dacwag CS, Bedford MT, Sif S, Imbalzano AN (2009) Distinct protein arginine methyltransferases promote ATP-dependent chromatin remodeling function at different stages of skeletal muscle differentiation. Mol Cell Biol 29(7):1909–1921. doi:10. 1128/mcb.00742-08 85. Mallappa C, Hu YJ, Shamulailatpam P, Tae S, Sif S, Imbalzano AN (2011) The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4. Nucleic Acids Res 39(4):1243–1255. doi:10. 1093/nar/gkq896 86. Gkountela S, Li Z, Chin CJ, Lee SA, Clark AT (2014) PRMT5 is required for human embryonic stem cell proliferation but not pluripotency. Stem cell Rev 10(2):230–239. doi:10.1007/ s12015-013-9490-z 87. Nicklay JJ, Shechter D, Chitta RK, Garcia BA, Shabanowitz J, Allis CD, Hunt DF (2009) Analysis of histones in Xenopus laevis. II. Mass spectrometry reveals an index of cell typespecific modifications on H3 and H4. J Biol Chem 284(2):1075–1085. doi:10.1074/jbc.M807274200 88. Shechter D, Nicklay JJ, Chitta RK, Shabanowitz J, Hunt DF, Allis CD (2009) Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions. J Biol Chem 284(2):1064–1074. doi:10.1074/jbc. M807273200 89. Wang WL, Anderson LC, Nicklay JJ, Chen H, Gamble MJ, Shabanowitz J, Hunt DF, Shechter D (2014) Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development. Epigenetics Chromatin 7:22. doi:10.1186/1756-8935-7-22 90. Chittka A, Nitarska J, Grazini U, Richardson WD (2012) Transcription factor positive regulatory domain 4 (PRDM4) recruits protein arginine methyltransferase 5 (PRMT5) to mediate histone arginine methylation and control neural stem cell proliferation and differentiation. J Biol Chem 287(51):42995–43006. doi:10.1074/jbc.M112.392746 91. Huang J, Vogel G, Yu Z, Almazan G, Richard S (2011) Type II arginine methyltransferase PRMT5 regulates the gene expression of inhibitors of differentiation/DNA binding ID2 and ID4 123 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. during glial cell differentiation. J Biol Chem 286:44429–44432. doi:10.1074/jbc.M111.277046 Kanade SR, Eckert RL (2012) Protein arginine methyltransferase 5 (PRMT5) signaling suppresses protein kinase Cdeltaand p38delta-dependent signaling and keratinocyte differentiation. J Biol Chem 287(10):7313–7323. doi:10.1074/jbc.M111. 331660 Paul C, Sardet C, Fabbrizio E (2012) The histone- and PRMT5associated protein COPR5 is required for myogenic differentiation. Cell Death Differ 19(5):900–908. doi:10.1038/cdd.2011. 193 Batut J, Duboe C, Vandel L (2011) The methyltransferases PRMT4/CARM1 and PRMT5 control differentially myogenesis in zebrafish. PLoS ONE 6(10):e25427. doi:10.1371/journal. pone.0025427 Kirino Y, Vourekas A, Sayed N, de Lima Alves F, Thomson T, Lasko P, Rappsilber J, Jongens TA, Mourelatos Z (2010) Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization. RNA 16(1):70–78. doi:10.1261/rna. 1869710 Nishida KM, Okada TN, Kawamura T, Mituyama T, Kawamura Y, Inagaki S, Huang H, Chen D, Kodama T, Siomi H, Siomi MC (2009) Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines. EMBO J 28(24):3820–3831. doi:10.1038/emboj.2009.365 Kirino Y, Kim N, de Planell-Saguer M, Khandros E, Chiorean S, Klein PS, Rigoutsos I, Jongens TA, Mourelatos Z (2009) Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability. Nat Cell Biol 11(5):652–658. doi:10.1038/ncb1872 Vagin VV, Wohlschlegel J, Qu J, Jonsson Z, Huang X, Chuma S, Girard A, Sachidanandam R, Hannon GJ, Aravin AA (2009) Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members. Genes Dev 23(15):1749–1762. doi:10.1101/gad. 1814809 Reuter M, Chuma S, Tanaka T, Franz T, Stark A, Pillai RS (2009) Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile. Nat Struct Mol Biol 16(6):639–646. doi:10. 1038/nsmb.1615 Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi–piRNA pathway provides an adaptive defense in the transposon arms race. Science 318(5851):761–764. doi:10.1126/science.1146484 Siomi MC, Mannen T, Siomi H (2010) How does the royal family of Tudor rule the PIWI-interacting RNA pathway? Genes Dev 24(7):636–646. doi:10.1101/gad.1899210 Kirino Y, Vourekas A, Kim N, de Lima Alves F, Rappsilber J, Klein PS, Jongens TA, Mourelatos Z (2010) Arginine methylation of vasa protein is conserved across phyla. J Biol Chem 285(11):8148–8154. doi:10.1074/jbc.M109.089821 Kim S, Günesdogan U, Zylicz Jan J, Hackett Jamie A, Cougot D, Bao S, Lee C, Dietmann S, Allen George E, Sengupta R, Surani MA (2014) PRMT5 protects genomic integrity during global DNA demethylation in primordial germ cells and preimplantation embryos. Mol Cell. doi:10.1016/j.molcel.2014. 10.003 Ohinata Y, Payer B, O’Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A, Saitou M, Surani MA (2005) Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436(7048):207–213. doi:10.1038/nature03813 Durcova-Hills G, Tang F, Doody G, Tooze R, Surani MA (2008) Reprogramming primordial germ cells into pluripotent stem cells. PLoS ONE 3(10):e3531. doi:10.1371/journal.pone.0003531 The PRMT5 arginine methyltransferase 106. Hayashi K, de Sousa Lopes SM, Surani MA (2007) Germ cell specification in mice. Science 316(5823):394–396. doi:10.1126/ science.1137545 107. Dhar S, Vemulapalli V, Patananan AN, Huang GL, Di Lorenzo A, Richard S, Comb MJ, Guo A, Clarke SG, Bedford MT (2013) Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3:1311. doi:10.1038/srep01311 108. Guderian G, Peter C, Wiesner J, Sickmann A, Schulze-Osthoff K, Fischer U, Grimmler M (2011) RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity. J Biol Chem 286(3):1976–1986. doi:10.1074/jbc.M110.148486 109. Teng Y, Girvan AC, Casson LK, Pierce WM Jr, Qian M, Thomas SD, Bates PJ (2007) AS1411 alters the localization of a complex containing protein arginine methyltransferase 5 and nucleolin. Cancer Res 67(21):10491–10500. doi:10.1158/00085472.can-06-4206 110. Gurung B, Feng Z, Iwamoto DV, Thiel A, Jin G, Fan CM, Ng JM, Curran T, Hua X (2013) Menin epigenetically represses Hedgehog signaling in MEN1 tumor syndrome. Cancer Res 73(8):2650–2658. doi:10.1158/0008-5472.can-12-3158 111. Gurung B, Hua X (2013) Menin/PRMT5/hedgehog signaling: a potential target for the treatment of multiple endocrine neoplasia type 1 tumors. Epigenomics 5(5):469–471. doi:10.2217/epi.13. 47 112. Gurung B, Feng Z, Hua X (2013) Menin directly represses Gli1 expression independent of canonical Hedgehog signaling. Mol Cancer Res 11(10):1215–1222. doi:10.1158/1541-7786.mcr-130170 113. Pesiridis GS, Diamond E, Van Duyne GD (2009) Role of pICLn in methylation of Sm proteins by PRMT5. J Biol Chem 284(32):21347–21359. doi:10.1074/jbc.M109.015578 114. Lacroix M, El Messaoudi S, Rodier G, Le Cam A, Sardet C, Fabbrizio E (2008) The histone-binding protein COPR5 is required for nuclear functions of the protein arginine methyltransferase PRMT5. EMBO Rep 9(5):452–458. doi:10.1038/embor.2008.45 115. Karolchik D, Barber GP, Casper J, Clawson H, Cline MS, Diekhans M, Dreszer TR, Fujita PA, Guruvadoo L, Haeussler M, Harte RA, Heitner S, Hinrichs AS, Learned K, Lee BT, Li CH, Raney BJ, Rhead B, Rosenbloom KR, Sloan CA, Speir ML, Zweig AS, Haussler D, Kuhn RM, Kent WJ (2014) The UCSC Genome Browser database: 2014 update. Nucleic Acids Res 42(D1):D764–D770. doi:10.1093/nar/gkt1168 116. Feng Y, Wang J, Asher S, Hoang L, Guardiani C, Ivanov I, Zheng YG (2011) Histone H4 acetylation differentially modulates arginine methylation by an in cis mechanism. J Biol Chem 286(23):20323–20334. doi:10.1074/jbc.M110.207258 117. Liu F, Zhao X, Perna F, Wang L, Koppikar P, Abdel-Wahab O, Harr MW, Levine RL, Xu H, Tefferi A, Deblasio A, Hatlen M, Menendez S, Nimer SD (2011) JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation. Cancer Cell 19(2):283–294. doi:10.1016/j.ccr.2010.12.020 118. Aggarwal P, Vaites LP, Kim JK, Mellert H, Gurung B, Nakagawa H, Herlyn M, Hua X, Rustgi AK, McMahon SB, Diehl JA (2010) Nuclear cyclin D1/CDK4 kinase regulates CUL4 expression and triggers neoplastic growth via activation of the PRMT5 methyltransferase. Cancer Cell 18(4):329–340. doi:10. 1016/j.ccr.2010.08.012 119. Chen M, Yi B, Sun J (2014) Inhibition of cardiomyocyte hypertrophy by protein arginine methyltransferase 5. J Biol Chem 289(35):24325–24335. doi:10.1074/jbc.M114.577494 120. Hou Z, Peng H, Ayyanathan K, Yan KP, Langer EM, Longmore GD, Rauscher FJ 3rd (2008) The LIM protein AJUBA recruits 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. protein arginine methyltransferase 5 to mediate SNAIL-dependent transcriptional repression. Mol Cell Biol 28(10):3198–3207. doi:10.1128/mcb.01435-07 Rho J, Choi S, Seong YR, Cho WK, Kim SH, Im DS (2001) Prmt5, which forms distinct homo-oligomers, is a member of the protein-arginine methyltransferase family. J Biol Chem 276(14):11393–11401. doi:10.1074/jbc.M008660200 Pal S, Baiocchi RA, Byrd JC, Grever MR, Jacob ST, Sif S (2007) Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J 26(15):3558–3569. doi:10.1038/sj.emboj.7601794 Gu Z, Gao S, Zhang F, Wang Z, Ma W, Davis RE, Wang Z (2012) Protein arginine methyltransferase 5 is essential for growth of lung cancer cells. Biochem J 446(2):235–241. doi:10. 1042/bj20120768 Barrallo-Gimeno A, Nieto MA (2005) The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132(14):3151–3161. doi:10.1242/dev.01907 Liang JJ, Wang Z, Chiriboga L, Greco MA, Shapiro E, Huang H, Yang XJ, Huang J, Peng Y, Melamed J, Garabedian MJ, Lee P (2007) The expression and function of androgen receptor coactivator p44 and protein arginine methyltransferase 5 in the developing testis and testicular tumors. J Urol 177(5):1918–1922. doi:10.1016/j.juro.2007.01.017 Powers MA, Fay MM, Factor RE, Welm AL, Ullman KS (2011) Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res 71(16):5579–5587. doi:10.1158/00085472.can-11-0458 Eckert D, Biermann K, Nettersheim D, Gillis AJ, Steger K, Jack HM, Muller AM, Looijenga LH, Schorle H (2008) Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells, CIS/IGCNU and germ cell tumors. BMC Dev Biol 8:106. doi:10.1186/1471-213x-8-106 Bao X, Zhao S, Liu T, Liu Y, Liu Y, Yang X (2013) Overexpression of PRMT5 promotes tumor cell growth and is associated with poor disease prognosis in epithelial ovarian cancer. J Histochem Cytochem 61(3):206–217. doi:10.1369/ 0022155413475452 Nicholas C, Yang J, Peters SB, Bill MA, Baiocchi RA, Yan F, Sif S, Tae S, Gaudio E, Wu X, Grever MR, Young GS, Lesinski GB (2013) PRMT5 is upregulated in malignant and metastatic melanoma and regulates expression of MITF and p27(Kip1.). PLoS ONE 8(9):e74710. doi:10.1371/journal.pone.0074710 Chi P, Allis CD, Wang GG (2010) Covalent histone modifications–miswritten, misinterpreted and mis-erased in human cancers. Nat Rev Cancer 10(7):457–469. doi:10.1038/nrc2876 Sawan C, Herceg Z (2010) Histone modifications and cancer. Adv Genet 70:57–85. doi:10.1016/b978-0-12-380866-0.60003-4 Yang Y, Bedford MT (2013) Protein arginine methyltransferases and cancer. Nat Rev Cancer 13(1):37–50. doi:10.1038/nrc3409 Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, Sun Y, Jacobsen A, Sinha R, Larsson E, Cerami E, Sander C, Schultz N (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Science Signal 6(269):pl1. doi:10.1126/scisignal.2004088 Kim JM, Sohn HY, Yoon SY, Oh JH, Yang JO, Kim JH, Song KS, Rho SM, Yoo HS, Kim YS, Kim JG, Kim NS (2005) Identification of gastric cancer-related genes using a cDNA microarray containing novel expressed sequence tags expressed in gastric cancer cells. Clin Cancer Res 11(2 Pt 1):473–482 Wei TY, Juan CC, Hisa JY, Su LJ, Lee YC, Chou HY, Chen JM, Wu YC, Chiu SC, Hsu CP, Liu KL, Yu CT (2012) Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the 123 N. Stopa et al. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. phosphoinositide 3-kinase/AKT signaling cascade. Cancer Sci 103(9):1640–1650. doi:10.1111/j.1349-7006.2012.02367.x Uzdensky A, Demyanenko S, Bibov M, Sharifulina S, Kit O, Przhedetski Y, Pozdnyakova V (2014) Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin. Tumour Biol 35(8):8225–8233. doi:10. 1007/s13277-014-2098-3 Han X, Li R, Zhang W, Yang X, Wheeler CG, Friedman GK, Province P, Ding Q, You Z, Fathallah-Shaykh HM, Gillespie GY, Zhao X, King PH, Nabors LB (2014) Expression of PRMT5 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth in vitro. J Neurooncol 118(1):61–72. doi:10.1007/s11060-014-1419-0 Yan F, Alinari L, Lustberg ME, Katherine Martin L, CorderoNieves HM, Banasavadi-Siddegowda Y, Virk S, BarnholtzSloan J, Bell EH, Wojton J, Jacob NK, Chakravarti A, Nowicki MO, Wu X, Lapalombella R, Datta J, Yu B, Gordon K, Haseley A, Patton JT, Smith PL, Ryu J, Zhang X, Mo X, Marcucci G, Nuovo G, Kwon CH, Byrd JC, Chiocca EA, Li C, Sif S, Jacob S, Lawler S, Kaur B, Baiocchi RA (2014) Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma. Cancer Res 74(6):1752–1765. doi:10.1158/0008-5472.can-13-0884 Gyorffy B, Surowiak P, Budczies J, Lanczky A (2013) Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS ONE 8(12):e82241. doi:10.1371/journal.pone. 0082241 Ibrahim R, Matsubara D, Osman W, Morikawa T, Goto A, Morita S, Ishikawa S, Aburatani H, Takai D, Nakajima J, Fukayama M, Niki T, Murakami Y (2014) Expression of PRMT5 in lung adenocarcinoma and its significance in epithelial-mesenchymal transition. Hum Pathol 45(7):1397–1405. doi:10.1016/j.humpath.2014.02.013 Shilo K, Wu X, Sharma S, Welliver M, Duan W, VillalonaCalero M, Fukuoka J, Sif S, Baiocchi R, Hitchcock CL, Zhao W, Otterson GA (2013) Cellular localization of protein arginine methyltransferase-5 correlates with grade of lung tumors. Diagn Pathol 8:201. doi:10.1186/1746-1596-8-201 Wei TY, Hsia JY, Chiu SC, Su LJ, Juan CC, Lee YC, Chen JM, Chou HY, Huang JY, Huang HM, Yu CT (2014) Methylosome protein 50 promotes androgen- and estrogen-independent tumorigenesis. Cell Signal 26(12):2940–2950. doi:10.1016/j. cellsig.2014.09.014 Scoumanne A, Zhang J, Chen X (2009) PRMT5 is required for cell-cycle progression and p53 tumor suppressor function. Nucleic Acids Res 37(15):4965–4976. doi:10.1093/nar/gkp516 Zhang HT, Zhang D, Zha ZG (1839) Hu CD (2014) Transcriptional activation of PRMT5 by NF-Y is required for cell growth and negatively regulated by the PKC/c-Fos signaling in prostate cancer cells. Biochim Biophys Acta 11:1330–1340. doi:10.1016/j.bbagrm.2014.09.015 Hsu JM, Chen CT, Chou CK, Kuo HP, Li LY, Lin CY, Lee HJ, Wang YN, Liu M, Liao HW, Shi B, Lai CC, Bedford MT, Tsai CH, Hung MC (2011) Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFRmediated ERK activation. Nat Cell Biol 13(2):174–181. doi:10. 1038/ncb2158 Lim JH, Lee YM, Lee G, Choi YJ, Lim BO, Kim YJ, Choi DK, Park JW (2014) PRMT5 is essential for the eIF4E-mediated 50 cap dependent translation. Biochem Biophys Res Commun. doi:10.1016/j.bbrc.2014.09.033 De Benedetti A, Graff JR (2004) eIF-4E expression and its role in malignancies and metastases. Oncogene 23(18):3189–3199. doi:10.1038/sj.onc.1207545 123 148. Jia Y, Polunovsky V, Bitterman PB, Wagner CR (2012) Capdependent translation initiation factor eIF4E: an emerging anticancer drug target. Med Res Rev 32(4):786–814. doi:10.1002/ med.21260 149. Jiang W, Roemer ME, Newsham IF (2005) The tumor suppressor DAL-1/4.1B modulates protein arginine Nmethyltransferase 5 activity in a substrate-specific manner. Biochem Biophys Res Commun 329(2):522–530. doi:10.1016/j. bbrc.2005.01.153 150. Jiang W, Newsham IF (2006) The tumor suppressor DAL-1/ 4.1B and protein methylation cooperate in inducing apoptosis in MCF-7 breast cancer cells. Mol Cancer 5:4. doi:10.1186/14764598-5-4 151. Fay MM, Clegg JM, Uchida KA, Powers MA, Ullman KS (2014) Enhanced arginine methylation of programmed cell death 4 protein during nutrient deprivation promotes tumor cell viability. J Biol Chem 289(25):17541–17552. doi:10.1074/jbc. M113.541300 152. Fisk JC, Read LK (2011) Protein arginine methylation in parasitic protozoa. Eukaryot Cell 10(8):1013–1022. doi:10.1128/ec. 05103-11 153. Liu CD, Cheng CP, Fang JS, Chen LC, Zhao B, Kieff E, Peng CW (2013) Modulation of Epstein–Barr virus nuclear antigen 2-dependent transcription by protein arginine methyltransferase 5. Biochem Biophys Res Commun 430(3):1097–1102. doi:10. 1016/j.bbrc.2012.12.032 154. Shire K, Kapoor P, Jiang K, Hing MN, Sivachandran N, Nguyen T, Frappier L (2006) Regulation of the EBNA1 Epstein–Barr virus protein by serine phosphorylation and arginine methylation. J Virol 80(11):5261–5272. doi:10.1128/jvi.02682-05 155. Doueiri R, Anupam R, Kvaratskhelia M, Green KB, Lairmore MD, Green PL (2012) Comparative host protein interactions with HTLV-1 p30 and HTLV-2 p28: insights into difference in pathobiology of human retroviruses. Retrovirology 9:64. doi:10. 1186/1742-4690-9-64 156. Boulanger MC, Liang C, Russell RS, Lin R, Bedford MT, Wainberg MA, Richard S (2005) Methylation of Tat by PRMT6 regulates human immunodeficiency virus type 1 gene expression. J Virol 79(1):124–131. doi:10.1128/jvi.79.1.124-131.2005 157. Beltran-Alvarez P, Espejo A, Schmauder R, Beltran C, Mrowka R, Linke T, Batlle M, Perez-Villa F, Perez GJ, Scornik FS, Benndorf K, Pagans S, Zimmer T, Brugada R (2013) Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel. FEBS Lett 587(19):3159–3165. doi:10.1016/j.febslet.2013.07.043 158. Yost JM, Korboukh I, Liu F, Gao C, Jin J (2011) Targets in epigenetics: inhibiting the methyl writers of the histone code. Curr Chem Genomics 5(Suppl 1):72–84. doi:10.2174/ 1875397301005010072 159. Copeland RA, Solomon ME, Richon VM (2009) Protein methyltransferases as a target class for drug discovery. Nat Rev Drug Discov 8(9):724–732. doi:10.1038/nrd2974 160. Daigle SR, Olhava EJ, Therkelsen CA, Majer CR, Sneeringer CJ, Song J, Johnston LD, Scott MP, Smith JJ, Xiao Y, Jin L, Kuntz KW, Chesworth R, Moyer MP, Bernt KM, Tseng JC, Kung AL, Armstrong SA, Copeland RA, Richon VM, Pollock RM (2011) Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell 20(1):53–65. doi:10.1016/j.ccr.2011.06.009 161. Yu W, Chory EJ, Wernimont AK, Tempel W, Scopton A, Federation A, Marineau JJ, Qi J, Barsyte-Lovejoy D, Yi J, Marcellus R, Iacob RE, Engen JR, Griffin C, Aman A, Wienholds E, Li F, Pineda J, Estiu G, Shatseva T, Hajian T, Al-awar R, Dick JE, Vedadi M, Brown PJ, Arrowsmith CH, Bradner JE, Schapira M (2012) Catalytic site remodelling of the DOT1L The PRMT5 arginine methyltransferase 162. 163. 164. 165. 166. 167. 168. methyltransferase by selective inhibitors. Nat Commun 3:1288. doi:10.1038/ncomms2304 Dhar SS, Lee SH, Kan PY, Voigt P, Ma L, Shi X, Reinberg D, Lee MG (2012) Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4. Genes Dev 26(24):2749–2762. doi:10. 1101/gad.203356.112 Tsai WW, Niessen S, Goebel N, Yates JR 3rd, Guccione E, Montminy M (2013) PRMT5 modulates the metabolic response to fasting signals. Proc Natl Acad Sci USA 110(22):8870–8875. doi:10.1073/pnas.1304602110 Tae S, Karkhanis V, Velasco K, Yaneva M, Erdjument-Bromage H, Tempst P, Sif S (2011) Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes. Nucleic Acids Res 39(13):5424–5438. doi:10.1093/nar/gkr170 Neuenkirchen N, Chari A, Fischer U (2008) Deciphering the assembly pathway of Sm-class U snRNPs. FEBS Lett 582(14):1997–2003. doi:10.1016/j.febslet.2008.03.009 Gonsalvez GB, Rajendra TK, Tian L, Matera AG (2006) The Sm-protein methyltransferase, dart5, is essential for germ-cell specification and maintenance. Curr Biol 16(11):1077–1089. doi:10.1016/j.cub.2006.04.037 Friesen WJ, Wyce A, Paushkin S, Abel L, Rappsilber J, Mann M, Dreyfuss G (2002) A novel WD repeat protein component of the methylosome binds Sm proteins. J Biol Chem 277(10):8243–8247. doi:10.1074/jbc.M109984200 Anne J, Ollo R, Ephrussi A, Mechler BM (2007) Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila. Development 134(1):137–146. doi:10.1242/dev. 02687 169. Li Z, Yu J, Hosohama L, Nee K, Gkountela S, Chaudhari S, Cass AA, Xiao X, Clark AT (2014) The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice. EMBO J. doi:10.15252/embj.201489319 170. Ren J, Wang Y, Liang Y, Zhang Y, Bao S, Xu Z (2010) Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis. J Biol Chem 285(17):12695–12705. doi:10.1074/jbc.M110.103911 171. Guo Z, Zheng L, Xu H, Dai H, Zhou M, Pascua MR, Chen QM, Shen B (2010) Methylation of FEN1 suppresses nearby phosphorylation and facilitates PCNA binding. Nat Chem Biol 6(10):766–773. doi:10.1038/nchembio.422 172. Wei TY, Hsia JY, Chiu SC, Su LJ, Juan CC, Lee YC, Chen JM, Chou HY, Huang JY, Huang HM, Yu CT (2014) Methylosome protein 50 promotes androgen- and estrogen-independent tumorigenesis. Cell Signal. doi:10.1016/j.cellsig.2014.09.014 173. Anne J, Mechler BM (2005) Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuleen. Development 132(9):2167–2177. doi:10.1242/dev.01809 174. Lacroix M, Messaoudi SE, Rodier G, Le Cam A, Sardet C, Fabbrizio E (2008) The histone-binding protein COPR5 is required for nuclear functions of the protein arginine methyltransferase PRMT5. EMBO Rep 9(5):452–458. http://www.nature.com/ embor/journal/v9/n5/suppinfo/embor200845_S1.html 175. Nishioka K, Reinberg D (2003) Methods and tips for the purification of human histone methyltransferases. Methods (San Diego, Calif) 31(1):49–58 pii: S1046202303000872 123 View publication stats