Arginine Methylation: The Coming of Age

Molecular Cell

Review Arginine Methylation: The Coming of Age Rome´o S. Blanc1,2 and Ste´phane Richard1,2,* 1Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montre´al, QC H3T 1E2, Canada 2Departments of Oncology and Medicine, McGill University, Montre ´ al, QC H2W 1S6, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.11.003

Arginine methylation is a common post-translational modification functioning as an epigenetic regulator of transcription and playing key roles in pre-mRNA splicing, DNA damage signaling, mRNA translation, cell signaling, and cell fate decision. Recently, a wealth of studies using transgenic mouse models and selective PRMT inhibitors helped define physiological roles for protein arginine methyltransferases (PRMTs) linking them to diseases such as cancer and metabolic, neurodegenerative, and muscular disorders. This review describes the recent molecular advances that have been uncovered in normal and diseased mammalian cells. Arginine Methylation and Demethylation Post-translational modifications (PTMs) lie at the heart of the fields of epigenetics and signal transduction. PTMs are involved in nearly all signaling cascades, often initiating or amplifying dynamic signals, which can be finely tuned to accommodate cellular requirements. The post-translation modification of histones as well as DNA methylation are key events of epigenetics influencing gene expression. Therefore, the enzymes that deposit and remove PTMs and DNA methylation are prime drug targets to alter specific pathways or gene expression and to treat certain diseases, including cancer. Arginine methylation is gaining traction as a key PTM, largely due to the generation of antibodies capable of detecting methylarginines, advanced proteomic techniques, small molecule inhibitors of protein arginine methyltransferases (PRMTs), and new transgenic animals to model human disease. With the interesting possibility that arginine demethylases exist, further roles of arginine methylation will soon be uncovered. The goal of this review is to describe the recent molecular advances that are regulated by arginine methylation. Protein Arginine Methylation The nitrogen atoms of arginine within polypeptides can be modified to contain methyl groups, a process termed arginine methylation. In mammals, arginine methylation is a modification as common as phosphorylation and ubiquitination (Larsen et al., 2016). It is carried out by the nine members of the PRMT family (Bedford and Clarke, 2009), although others may exist, such as the putative arginine methyltransferase NDUFAF7 (Zurita Rendo´n et al., 2014). PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the guanidino nitrogen atoms of arginine. This reaction results in the formation of methylarginine and S-adenosylhomocysteine. There are three main forms of methylarginines identified in eukaryotes: u-NG-monomethylarginine (MMA), u-NG,NG-asymmetric dimethylarginine (aDMA), and u-NG,N’G-symmetric dimethylarginine (sDMA) (Figure 1). PRMTs fall into three categories according to their catalytic activity; type I (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8) and type II (PRMT5 and PRMT9) enzymes carry out the formation of MMA as an intermediate before the establish8 Molecular Cell 65, January 5, 2017 ª 2017 Elsevier Inc.

ment of aDMA or sDMA, respectively (Yang et al., 2015). PRMT7 is a type III enzyme that catalyzes only the formation of MMA, and thus far, histones are its only known substrates (Feng et al., 2013). Arginine methylation is known to play a major role in gene regulation because of the ability of the PRMTs to deposit key activating (histone H4R3me2a, H3R2me2s, H3R17me2a, H3R26me2a) or repressive (H3R2me2a, H3R8me2a, H3R8me2s, H4R3me2s) histone marks. In addition, there are many substrates that are nonhistones involved in biological processes including transcription, cell signaling, mRNA translation, DNA damage signaling, receptor trafficking, protein stability, and pre-mRNA splicing (Auclair and Richard, 2013; Yang and Bedford, 2013). Arginine- and glycinerich motifs, termed RGG/RG motifs, are among the most common amino acid sequences favored by PRMTs, and these motifs often function in both nucleic acid binding and mediating protein-protein interactions (Thandapani et al., 2013). The presence of glycine neighboring arginine is predicted to enhance conformational flexibility and facilitate the access of the arginine to the active site of PRMTs. Not all PRMTs methylate RGG/RG motifs. CARM1/ PRMT4 prefers arginine neighboring a PGM-rich (proline, glycine, and methionine) motif (Yang and Bedford, 2013), while PRMT7 prefers RxR motifs surrounded by a lysine-rich environment (Feng et al., 2013). The main consequence of arginine methylation is alteration of its binding interactions. Given the nature of arginine with its five potential hydrogen-bond donors, the steric effects from adding a methyl group will affect interactions with the hydrogen-bond acceptors of their interacting partners without changing the charge (Fuhrmann et al., 2015). Methylarginine Interactors The methylation of arginines enhances interactions with Tudor domains (Gayatri and Bedford, 2014). The Tudor domains of SMN (Survival of motor neuron), SPF30 (Splicing factor 30), and TDRD1/2/3/6/9/11 (Tudor domain-containing protein) are currently the main known methylarginine-interacting domains (Tripsianes et al., 2011). Tudor domains form an aromatic cage such that cation-p contacts occur between their aromatic residues and the cationic carbon of methylarginine. The methyl groups provide increased hydrophobicity within the Tudor

Molecular Cell

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CH2

Oxidized aDMA Intermediate

JmjC Family Fe(II)

* Asymmetrical Dimethyl Arginine (aDMA)

Arginine Monomethyl Arginine (MMA)

Symmetrical Dimethyl Arginine (sDMA)

Citrulline

Legend Favor : Type I enzyme Type II enzyme Type III enzyme Mutated enzyme

*RxR motifs **RGG/RG motifs *** Neighboring PGM motifs

Figure 1. Protein Arginine Methylation and Demethylation Type I, II, and III protein arginine methyltransferases (PRMTs) generate monomethylarginine as a first step, followed by asymmetrical dimethylation (aDMA; Type I) and symmetrical dimethylation (sDMA; Type II) on the nitrogen atoms of the guanidino group. PRMT mutants can exhibit a different activity than their primary type (PRMT9C431H, PRMT1 M155A/M48L, PRMT7E181D, and PRMT7E181D/Q329A). Asterisks indicate favored substrate motif. Arginine demethylation could be done by Jumonji C domain containing proteins (JmJC) which catalyze demethylation in an oxidative fashion.

domain binding pocket. There are 36 proteins that harbor at least one Tudor domain in humans compared to >2,000 proteins containing RGG/RG motifs, suggesting that other methylargi-

nine-interacting domains are likely to be discovered. Tudor domains as methylarginine interactors were recently reviewed (Gayatri and Bedford, 2014).

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Molecular Cell

Review Protein Arginine Demethylation The existence of enzymes capable of demethylating methylarginines is central to the concept that arginine methylation is a dynamic modification. The existence of arginine demethylases is controversial (Yang and Bedford, 2013). A putative arginine demethylase, JmjD6, was identified but later shown to be a lysine hydroxylase (Webby et al., 2009). Recently, it was shown that certain lysine demethylases (KDM3A, KDM4E, KDM5C) also possess arginine demethylation activity in vitro (Walport et al., 2016). Since the enzymatic mode of action for arginine demethylation is similar to lysine demethylation, it seems likely that other JmjC proteins will also demethylate methylarginine. Further investigation is required to confirm the function of these dual lysine/arginine demethylases in vivo and to explore how these two different activities interplay. Protein Arginine Methyltransferases Overview: PRMT Activity and Regulation The PRMT structures reveal the presence of seven b strand catalytic domains with variations in their structure that make them unique. Type I and II enzymes have a central cavity and two opposing active sites in their head-to-tail homodimer structure (Zhang and Cheng, 2003). These active sites contain a highly conserved SAM binding pocket characterized by the presence of an E-loop critical for substrate recognition and methylation. PRMT5 is unique in that it requires the methylosome protein 50 (MEP50/WDR77) to form an active enzymatic complex, and this interaction defines its distributive mode of action (Antonysamy et al., 2012). Type III enzyme PRMT7 is an exception, as it lacks the central cavity and acts as a monomer that acquires a homodimer-like structure with two catalytic domains both essential for its activity. PRMT7 also requires the E-loop, which is critical for preserving its unique type III activity (Debler et al., 2016; Jain et al., 2016). Type I. PRMT1 is the main enzyme responsible for the generation of aDMA in proteins, and it has a preference for RGG/RG motifs (Thandapani et al., 2013); however, there are numerous exceptions. PRMT1 functions as a transcriptional co-activator by depositing dimethylarginines on H4R3 (Bedford and Clarke, 2009). PRMT1 also methylates RNA binding and DNA damage proteins to modulate RNA metabolism and maintain genome stability, respectively (Auclair and Richard, 2013). PRMT1 was shown to homodimerize (Zhang and Cheng, 2003), and its activity to be highly dependent on, two methionine residues, M48 and M155, which alter substrate specificity and abolish catalytic activity if mutated (Gui et al., 2014). Interestingly, loss of PRMT1 activity increases MMA- and sDMA-proteins owing to substrate scavenging by other PRMTs (Dhar et al., 2013). PRMT1 is regulated by alternative splicing resulting in seven different isoforms, suggesting that each isoform with its distinctive N terminus has unique substrate preference (Goulet et al., 2007). PRMT1 is regulated by oxidation (Morales et al., 2015) and microRNAs including miR503 (Li et al., 2015a). PRMT2 has been shown to function as a transcriptional repressor (Ganesh et al., 2006). Notably, PRMT2 expression increases in hypoxic conditions (Yildirim et al., 2006) and decreases in elevated glucose conditions. PRMT3 is unique among PRMTs for its zinc-finger at its N terminus, which confers substrate specificity (Frankel and Clarke,

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2000). It methylates ribosomal proteins (Swiercz et al., 2007), and its activity is regulated by interacting proteins such as DAL-1/4.1B (Singh et al., 2004). CARM1/PRMT4 is known for its transcription coactivator function. It methylates H3R17 and H3R26 and also potentiates transcription by methylating and recruiting transcription factors directly. CARM1 activity is regulated by association with CBP/p300 acetyltransferase and UPF1. CBP-mediated acetylation of histone H3K18, and subsequently at K23, is sufficient to recruit CARM1 to methylate H3R17 (Bedford and Clarke, 2009). CARM1 also regulates nonsense-mediated mRNA decay by associating with UPF1 and recruiting it to premature terminating codon-containing transcripts (Sanchez et al., 2016). CARM1 is involved in regulating pre-mRNA splicing (Cheng et al., 2007; Kuhn et al., 2011). Its expression is regulated by various microRNAs including miR181c (Xu et al., 2013), miR-223 (Vu et al., 2013), and miR-15 (Liu et al., 2014). CARM1 automethylation and O-linked-b-Nacetylglucosaminidation determines substrate specificity (Charoensuksai et al., 2015). PRMT6 is predominantly nuclear and methylates RGG/RG motif; however, it also methylates arginines neighboring charged residues as observed with HIV Tat (Boulanger et al., 2005). PRMT6-mediated methylation is generally associated with transcriptional repression by generating H3R2me2a (Neault et al., 2012). PRMT6 automethylation increases its stability (Singhroy et al., 2013). Brain-specific PRMT8 is membrane-bound type I arginine methyltransferase. X-ray crystallography shows it forms a tetrameric structure for substrate recognition and specificity (Lee et al., 2005; Lee et al., 2015). PRMT8 also has been reported to harbor noncanonical phospholipase D activity (Kim et al., 2015b). Type II. In mammals, PRMT5 is the main type II enzyme responsible for the majority of sDMA formation in polypeptides. It methylates in a distributive, rather than processive, manner (Wang et al., 2014b), meaning PRMT5 releases MMA before the second methylation event. In vitro, PRMT5 activity requires the formation of a hetero-octameric complex with MEP50, which mediates substrate specificity and interaction with binding partners (Antonysamy et al., 2012). Although MEP50 is required to activate PRMT5 for substrate recognition in vitro (Wang et al., 2014b), the absolute requirement of MEP50 for PRMT5 function in vivo remains unclear (Tee et al., 2010). PRMT5 interaction with various partners, including plCln, and the kinase RioK1, is important to regulate its catalytic activity. These partners bind PRMT5 in a mutually exclusive manner to control substrate specificity (Guderian et al., 2011). As a cautionary note, anti-FLAG antibodies are known to non-specifically immunopurify PRMT5 (Nishioka and Reinberg, 2003). Besides methylating histones and functioning, in general, as a transcriptional corepressor, PRMT5 methylates Sm proteins for their assembly into mature small nuclear ribonucleoproteins (snRNPs) (Meister et al., 2001). Thus, it is not surprising that PRMT5-deficient cells harbor numerous defects in splicing (Bezzi et al., 2013). PRMT9 has recently been shown to be a type II enzyme that is not redundant with PRMT5 (Hadjikyriacou et al., 2015; Yang et al., 2015). PRMT9 methylates spliceosome-associated protein 145 (SAP145) to regulate alternative splicing (Yang et al., 2015). PRMT9, like PRMT7, contains two catalytic domains with conserved sequences in the double E-loop important for

Molecular Cell

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Repression

Legend PotenƟate Repress MMA

Me

aDMA sDMA 3-Me Acetyl

Me

Me

Me

Ac

TF

Reader

CF

Transcription Factor

Methylation Reader

Co-factor

Figure 2. Arginine Methylation at the Crossroads of Transcriptional Regulation Arginine methylation potentiates (green arrow) or represses (red arrow) interaction with other factors (readers) and, subsequently, other posttranslational modifications (lysine methylation and acetylation). These cross-talks regulate the activation (left) or repression (right) of transcription via enzymes that catalyze methylation on histones H3 (upper) and H4 (lower), as well as non-histone proteins involved in various signaling cascades.

substrate specificity and activity (Hadjikyriacou et al., 2015; Jain et al., 2016). Mutations within the catalytic domain can switch PRMT9 activity from a type II to a type III enzyme (Figure 1) (Jain et al., 2016). Type III. PRMT7 plays a role in transcriptional regulation, snRNP biogenesis, and splicing. PRMT7 was first identified in a screen where its depletion leads to increase sensitivity to topoisomerase II inhibitors (Gros et al., 2006). Although the mechanism of action of PRMT7 in DNA damage signaling remains to be defined, it was shown that cells depleted of PRMT7 upregulate genes of the DNA repair machinery such as POLD1, POLD2, ALKBH5, and APEX2 (Karkhanis et al., 2012). The catalytic activity of PRMT7 is dependent on the highly conserved double E-loop containing two residues critical for substrate preference; mutating these residues switches PRMT7 to a type I or II enzyme (Figure 1) (Debler et al., 2016; Jain et al., 2016). Presently, the relationship between type III and type I and II enzymes is not understood, and there is no evidence of priming by PRMT7 monomethylation for subsequent dimethylation by type I and II enzymes. Moreover, the substrate sequence preference of PRMT7 (RxR) is different from that of main type I (PRMT1) and type II (PRMT5) enzymes. Several other observations that are unresolved include the fact that H3R2me2s (Migliori et al., 2012) and H4R3me2s marks (Blanc et al., 2016a) are decreased in PRMT7-deficient cells. Transcriptional Regulation by Arginine Methylation While PRMTs are known regulators of transcription, the mechanistic details underlying the recruitment of methylreading coactivator and corepressor complexes functioning with the basal transcription machinery requires further investigation.

Histones. Activator. The main enzymes that function as coactivators are PRMT1 and CARM1 (Figure 2). The methylation of H4R3me2a by PRMT1 is generally associated with active transcription, as it recruits the p300/CBP-associated factor complex and potentiates acetylation of histone H3 at lysines 9 and 14, two known active modifications facilitating the binding of transcription factors (Bedford and Clarke, 2009). This crosstalk between H4R3me2a and H3K9/14Ac was reported to be present in various biological processes from cell differentiation to the response to cocaine (Li et al., 2015c). CARM1 is a transcriptional coactivator that preferentially methylates H3R17, H3R26, and H3R42. The resulting H3R17/R26/R42me2a marks are associated with activation of transcription. CARM1-mediated methylation of histone H3 is also associated with reduced binding of the nucleosome remodeling and deacetylase complex (Yang and Bedford, 2013). Repressor. The major enzymes that function as corepressors are PRMT5 and PRMT6 (Figure 2). PRMT5 methylates H4R3me2s and H3R8me2s, while PRMT6 methylates H3R2me2a. The latter mark antagonizes the neighboring H3K4me3 activation mark by preventing the binding of the Mixed Lineage Leukemia (MLL) complex (Guccione et al., 2007; Neault et al., 2012). PRMT6 repressive activity can also occur via the methylation of H2AR29me2a (Waldmann et al., 2011). PRMT6 also activates transcription, like CARM1, through methylation of H3R42 (Casadio et al., 2013). The PRMT5-mediated methylation mark, H4R3me2s, is recognized by the DNA methyltransferase DNMT3a. Once recruited, DNMT3a methylates neighboring DNA to further

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Review strengthen the associated gene repression (Zhao et al., 2009). PRMT5 can also potentiate transcription through methylation of H3R2me2s preventing recruitment of repressor complexes, enhancing binding of the transcriptional co-activator via WDR5 (Migliori et al., 2012). Non-histones. PRMT1 also positively regulates transcription via direct methylation of Ash2L, a protein subunit of the H3K4 methyltransferase complexes (Butler et al., 2011) (Figure 2). PRMT1 regulates TNFa/NF-kB signaling via methylation of RelA R30, which prevents it binding to DNA (Reintjes et al., 2016). CARM1 with the acetyltransferase p300 regulate NF-kB recruitment and transcriptional activity (Covic et al., 2005) (Figure 2). Arginine methylation of non-histone proteins is also involved in other biological processes, which were widely discussed by Wei and colleagues (2014) in the context of disease and by Biggar and Li (2015) in the context of cellular signaling (Biggar and Li, 2015). Arginine Methylation Roles In Vivo Full-Body Knockout Over the last decade, extensive efforts have been concentrated on identifying the physiological roles of arginine methylation. The earliest known epigenetic mark that contributes to the inner cell mass is arginine methylation of histone H3 (Torres-Padilla et al., 2007), perfectly illustrating the importance of this post-translational modification during development. With recent technological advances, transgenic mouse models and genome-wide studies have become easily accessible and widely used. Specifically, conditional PRMT alleles provide in vivo animal models to study tissue-specific roles of arginine methylation during development, adult homeostasis, aging, and cancer (Figures 3, 4, and 5). As PRMTs are frequently overexpressed in cancer, gain-of-function transgenic alleles of PRMTs have also been generated and contribute to our understanding of cancer initiation and progression (Di Lorenzo et al., 2014). Full-body deletion of PRMT1 or PRMT5 is, unsurprisingly, embryonic lethal (Pawlak et al., 2000; Tee et al., 2010; Yu et al., 2009). The loss of PRMT1 in mouse embryonic fibroblasts (MEFs) induces a halt in proliferation with remarkable genomic instability (Yu et al., 2009). PRMT5 is also critical for the maintenance of pluripotency in mouse embryonic stem cells (ESCs), as it represses key differentiation genes (Tee et al., 2010). Conversely, in human ESCs, PRMT5 is required for proliferation but dispensable for pluripotency (Gkountela et al., 2014). Three independent transgenic PRMT7 alleles exist, and the viability of full body PRMT7 null mice depends on the allele and genetic background. Prmt7tm1a(EUCOMM) was reported to generate non-Mendelian ratios at birth with features resembling human PRMT7 mutation carriers (Akawi et al., 2015). In a C57BL/6 background, Prmt7tm1a(EUCOMM) displayed no overt phenotype except for late-onset obesity. It is not clear whether Prmt7tm1a(EUCOMM) is a complete null or a hypomorphic allele, as there appears to remain a truncated form of PRMT7 (Jeong et al., 2016). Ying et al. (2015) reported that their mouse model targeting exon 5 produced in a mixed C57BL/6-129sv background was born in Mendelian ratios without overt phenotype but died at 5–10 post-natal days (Ying et al., 2015). PRMT7 null mice targeting exon 4 in a pure C57BL/6 background display

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early post-natal developmental delays characterized by short stature, which is then rectified in adulthood, as well as signs of obesity and premature aging characterized by loss of skeletal muscle (Blanc et al., 2016a). Full-body CARM1 knockout mice are born smaller than their wild-type littermates and die shortly after birth. CARM1 is required for the epigenetic maintenance of pluripotency and self-renewal, as it methylates H3R17 and H3R26 at core pluripotency genes such as Oct4, Sox2, and Nanog (Torres-Padilla et al., 2007; Wu et al., 2009). CARM1 also methylates Sox2 R113 leading to self-association and transactivation (Zhao et al., 2011). CARM1-mediated methylation was also described as a tuning mechanism to regulate the amplitude and duration of Notch1 response by methylating its intracellular domain (Hein et al., 2015). These findings define a role for CARM1 in cell signaling contributing to differentiation. Interestingly, the generation of a CARM1 catalytically dead mutant mouse model demonstrates that its activity is critical for the vast majority of its in vivo functions such as cell differentiation, embryogenesis, and gene co-activation (Kim et al., 2010). It was shown that repression of CARM1 by miR181c promotes differentiation of hESC (Xu et al., 2013). CARM1 was recently found to be involved in the regulation of Sox21 expression during cell fate decisions (Goolam et al., 2016). Mice null for PRMT2, PRMT3, PRMT6, or PRMT8 are viable. PRMT2 null mice are lean and resistant to dietary-induced obesity. They have reduced serum leptin levels and are sensitive to exogenous leptin administration (Iwasaki et al., 2010). Importantly, PRMT2 was shown to mediate the dorsal developmental program through methylation of H3R8me2a (Blythe et al., 2010). It was further suggested that this PRMT2-associated mark is co-associated with the active mark H3K4me3 to mediate the Spindlin1-Wnt/b-Catenin signaling axis at the transcriptional level (Su et al., 2014), critical for both embryonic development and adult homeostasis. Like PRMT7, PRMT3 null mice harbor a developmental delay resulting in a small size after birth, but a normal adult size (Swiercz et al., 2007). However, adult PRMT3 have Minute-like characteristics mimicking the phenotype of rpS2 mutant Drosophila, a well-known PRMT3 substrate (Swiercz et al., 2007). PRMT6 null mice do not exhibit obvious macroscopic defects (Neault et al., 2012). The generation of a gain-of-function mouse model shows that PRMT6 co-activates NF-kB (Di Lorenzo et al., 2014). In Zebrafish, PRMT6 is required for early development, as loss of function results in early epiboly defects due to p38/JNK signaling activation and apoptosis (Zhao et al., 2016b). PRMT6-mediated histone methylation regulates poised chromatin to maintain the balance between pluripotency and differentiation, regulating ESC fate (Lee et al., 2012). The role of PRMT6 in cell proliferation and regulation of cellular senescence suggests that, like PRMT7, it may play a role in aging (Blanc et al., 2016a; Neault et al., 2012). The expression of PRMT8 is induced during mouse ESC differentiation into neural progenitors (Solari et al., 2016). Nervous System Neural Stem Cells. In neural stem cells, Schwann Cell factor 1 (SC1) recruits PRMT5 to chromatin to epigenetically modulate neural stemness by regulating self-renewal capacity and cellcycle progression (Chittka et al., 2012) (Figure 3). PRMT5 is

Molecular Cell

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PRMT1 depletion in the CNS is characterized by death at postnatal week 2.

(Huang et al., 2012)

In mouse, primary oligodendrocyte progenitors, PRMT5 represses Id2 and Id4, two repressors of glial differentiation

Figure 3. Arginine Methylation Regulates Neural Stem Cell Fate during Embryogenesis The absence of the mentioned PRTMs in the central nervous system (CNS; Nestin-cre), primary neural stem cell, or progenitors reveals that arginine methylation is critical for the establishment or the maintenance of neural lineages during embryogenesis.

required for neural stem cell survival, and its depletion in the central nervous system (CNS) with the Nestin-Cre transgenic mouse results in CNS developmental defects and lethality within 14 days after birth (Bezzi et al., 2013) (Figure 3). PRMT7 is also

implicated in neural differentiation, as it blocks MLL4-mediated differentiation in the human progenitor cell line, NT2/D1 (Dhar et al., 2012). The methylation of H4R3 prevents MLL4 binding and subsequent methylation of its target H3K4 mark. This

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Review resembles the Kabuki syndrome mutations in the PHD MLL4 domain, known to recognize H4R3me2a (Dhar et al., 2012) (Figure 3). Glial Lineage. PRMT1 is essential for the development of CNS myelination. PRMT1 specific depletion in the CNS using NestinCre leads to death at the second post-natal week. The mice have impaired oligodendrocyte differentiation, characterized by a decrease in mature myelin markers including myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), with no critical effects observed in neurons or astrocytes (Hashimoto et al., 2016) (Figure 3). PRMT5 was also shown to be required for oligodendrocyte differentiation, as its reduced expression increases expression of Id2 and Id4, repressors of differentiation, by influencing DNA methylation of their gene promoters (Huang et al., 2011) (Figure 3). CARM1-mediated methylation of H3R17 is required for both the establishment and the maintenance of the astroglial lineage. The absence of H3R17me2a downregulates miR-92a levels (Selvi et al., 2015), and this miRNA is known to participate in neural development (Bian et al., 2013). It was also reported that lack of this histone mark decreases levels of miR-10a and miR-575, which are suspected of maintaining the astroglial lineage (Cho et al., 2011), supporting the requirement of CARM1 for the establishment of this lineage (Figure 3). Neuronal Lineage. PRMT8 was shown to serve as a rheostat along with PRMT1 to regulate cell fate during retinoic acidinduced neuronal differentiation. Moreover, PRMT8 acts as a coactivator to drive late differentiation (Simandi et al., 2015). Deletion of PRMT8 in mice leads to impaired locomotor behavior associated with a tail-lifting clasping phenotype, reduced motor coordination, and hyperactivity. These defects were explained by alteration of the cerebellum structure due to a lack of the PRMT8 phospholipase D activity, which is critical for Purkinje neuron dendritic arborisation (Kim et al., 2015b). PRMT3 could also play a role in mouse motor functions. An immunohistochemical study in mouse brain revealed that PRMT3 is expressed solely in neuronal cell bodies and dendrites. Its uneven repartition in neurons suggests a specific function in the neuronal circuits associated with motor and limbic systems since higher expression was found in these areas (Ikenaka et al., 2006). It was further shown in rat hippocampal neurons that absence of PRMT3, and consequently decreased rpS2 protein stability, affects neuron spine shape, but not numbers, suggesting a role for PRMT3 in the neuronal translation machinery mediating dendritic spine activity (Miyata et al., 2010). In medium spiny neurons expressing the dopamine D2 receptors, PRMT6 and its associated repressive mark (H3R2me2a) decrease in response to chronic cocaine exposure leading to gene activation of Src kinase signaling inhibitor 1 (Srcin1), a suppressor of Src signaling (Damez-Werno et al., 2016). Muscular System The role of arginine methylation in skeletal muscle differentiation and regeneration has recently received lots of attention (Blanc et al., 2016a; Kawabe et al., 2012; Zhang et al., 2015). CARM1 is required for late stages of myogenesis, as it facilities chromatin-remodeling mediated by the SWI/SNF complex (Dacwag et al., 2009) and regulates the glycogen gene program (Wang et al., 2012). CARM1 conditional ablation in muscle stem cells

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(Pax7-Cre) reveals its role in the myogenic differentiation program to regulate asymmetric division in response to injury. Mechanistically, CARM1 methylates the transcription factor Pax7, resulting in recruitment of MLL1/2 and activation of Myf5 expression (Kawabe et al., 2012) (Figure 4). During myogenesis, PRMT5 plays a transcriptional role. Like CARM1, PRMT5 regulates myogenesis by facilitating SWI/ SNF-mediated chromatin remodeling. PRMT5 also associates with and methylates histones at the promoter of the differentiation factor myogenin and other myogenic program genes (Dacwag et al., 2009). In vivo, PRMT5 conditional ablation in muscle stem cells does not affect myogenesis but demonstrates that PRMT5 is required for the adult stem cell expansion during injury-induced regeneration. The absence of PRMT5 results in a p53-independent increase of p21 levels with associated premature fibrosis and loss of the muscle stem cell niche, defining a role for PRMT5 in long-term muscle stem cell maintenance (Zhang et al., 2015) (Figure 4). PRMT7 is highly expressed in skeletal muscle. Moreover, PRMT7 depletion in mice is associated with obesity and agerelated skeletal muscle loss (Blanc et al., 2016a; Jeong et al., 2016). Conditional depletion of PRMT7 in muscle stem cells (Pax7-Cre) does not affect resting muscle but impairs skeletal muscle regeneration upon injury (Blanc et al., 2016a). It was also reported that PRMT7 deficient muscles have decreased oxidative metabolism with decreased expression of genes involved in muscle oxidative metabolism (Jeong et al., 2016). The absence of PRMT7 leads to premature entrance into senescence with increased p21 levels in a p53-independent fashion. Elevated p21 expression is explained by the absence of DNMT3b and subsequent hypomethylation of CpG islands of the p21/CDKN1A gene promoter. Rapid entry of PRMT7 null muscle stem cells into senescence in vivo results in loss of proliferation, delay in differentiation, and reduction of the muscle stem cell pool, as it abolishes long-term self-renewal, ultimately impeding regeneration (Blanc et al., 2016a). Interestingly, the loss of muscle mass observed in aging PRMT7 null mice correlates with the decrease of PRMT7 expression in aging wildtype mice, suggesting PRMT7 could also play a role in muscle homeostasis and be a potential candidate to study in sarcopenia-associated diseases (Blanc et al., 2016a; Jeong et al., 2016) (Figure 4). PRMT1 is also required for adult skeletal muscle regeneration. PRMT1 methylates the Six1/4 coactivator Eya1 involved in myogenesis. The absence of PRMT1 in the Pax7-Cre mouse model leads to hypomethylation of Eya1 and subsequent decrease of Eya1 binding to the MyoD promoter. MyoD levels decrease in PRMT1 null muscle stem cells, impairing their differentiation, and increasing their expansion in vivo, ultimately leading to an increase of Pax7-positive cells with disrupted regeneration (Blanc et al., 2016b) (Figure 4). Immune System Arginine methylation plays a critical role in the establishment and maintenance of the lymphoid and myeloid lineages (Figure 5). Their critical role in inflammatory responses was recently discussed (Greenblatt et al., 2016; Kim et al., 2016). Hematopoietic Stem Cells and Progenitors. PRMTs also play an important role in the cell fate decisions of blood progenitors.

Molecular Cell

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Figure 4. PRMTs Regulate Adult Muscle Stem Cell Function The indicated PRMTs were shown to be required for muscle stem cell regenerative function in vivo using the Pax7-cre transgenic mouse model to deplete PRMT in muscle stem cells.

PRMT5 is essential in adult mice to maintain the pool of hematopoietic cells. Conditional ablation of PRMT5 (Mx1-Cre) in adult hematopoietic stem cells results in visible anemia, due to pancytopenia. On the short term, both the absence of PRMT5 and inhibition of its activity using an inhibitor promote HSC commitment but over time disrupt cell-cycle progression in progenitors, resulting in impaired cytokine-mediated signaling and increased p53 signaling. Taken together, these observations suggest that PRMT5 is required for the maintenance of HSC in quiescence, as well as for progenitor expansion. Lymphoid. PRMT1 is necessary for normal B cell development (Hata et al., 2016). This is at least, in part, explained by PRMT1

promoting B cell differentiation in mice by methylating R198 of the Iga chain of the B cell receptor (BCR). Methylation prevents activation of the phosphatidylinositol 3-kinase and calcium release downstream of the BCR, which are known negative regulators of differentiation (Infantino et al., 2010). Deleting PRMT5 in bone marrow cells reduced pro- and pre-B and impaired T cell development (Liu et al., 2015), suggesting that it is required for lymphocyte development. Finally, PRMT7 represses Bcl6 expression by influencing the presence of the H4R3me1/me2s repressive mark at Bcl6 gene promoter. PRMT7 conditional depletion in B cells results in decreased marginal zone B cell number, an increase in follicular cells, and anomalous germinal

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Figure 5. Immune System Needs PRMTs to Generate Mature Cells Depletion of indicated PRMT in hematopoietic stem cells or progenitors results in the loss of specific lineage and/or maturation in both the lymphoid and myeloid lineages.

center formation due to Bcl6 sustained expression (Ying et al., 2015). Reproductive System PRMT5 is required for mouse primordial germ cell survival, but not specification (Li et al., 2015d). At E7.5, the primordial germ cell specification transcription factor BLIMP1 associates with PRMT5, resulting in an increase of the PRMT5-associated marks H2A/H4R3me2s. The BLIMP1-PRMT5 complex is then translocated into the cytoplasm at E8.5, consequently decreasing the aforementioned histone marks. It was, however, shown that PRMT5 does not contribute to BLIMP1-mediated primordial germ cell specification, but instead ensures their survival and proliferation by methylating Sm proteins and regulating premRNA splicing (Li et al., 2015d). PRMT5-associated marks are also required to repress LINE1 and IAP transposons during the embryogenesis-associated DNA demethylation. This preserves the genomic integrity of primordial germ cells during their epigenetic reprogramming (Kim et al., 2014b). In androgen receptor null mice, PRMT6 is upregulated in spermatocytes. Thus the

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repression of PRMT6 in these mice leads to germ cell apoptosis, suggesting that in the absence of the androgen receptor, PRMT6 is required for their survival (Luo et al., 2015). PRMT7 was suggested to be involved in paternal imprinting, as it interacts with the CTCFL/BORIS transcription factor, resulting in altered histone methylation patterns and DNA methylation at the imprinting control region (ICR) of the H19/Igf2 locus (Jelinic et al., 2006). Reprogramming As described above, arginine methylation plays a critical role in stem cell maintenance and differentiation. It is also known to play a role in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. PRMT5, along with other original Yamanaka factors (OCT4, SOX2, KLF4, C-MYC [OSKM]), can act as a reprogramming factor (Nagamatsu et al., 2011). Depleting cells of PRMT5 reduced the number of Nanog-GFP positive iPS cells induced with OSKM (Nagamatsu et al., 2011). In dairy goat embryonic fibroblasts, expression of PRMT5 plus OSKM was later reported to significantly increase the number of alkaline phosphatase-positive iPS-like cells compared to

Molecular Cell

Review OSKM alone (Chu et al., 2015). PRMT5 enhances the generation of iPS cells by downregulating p53, p21, and caspase 3 signaling (Chu et al., 2015). PRMT5 was also shown to methylate KLF4, inhibiting its ubiquitination by VHL (Hu et al., 2015). PRMT7 is capable of replacing SOX2 in iPS generation, and it was identified in a factor-substitution assay (Wang et al., 2016). As PRMT5 also substitutes for SOX2 (Nagamatsu et al., 2011), it suggests that PRMT5, PRMT7, and SOX2 act in the same pathway to promote the generation of iPS cells. Conclusive Remarks The participation of PRMTs in stem cell biology is irrefutable. Overall, studies show that, in most cases, disruption of PRMTs results in the loss of specific lineages during development (Hashimoto et al., 2016; Selvi et al., 2015) and dysregulation of the balance between proliferation and differentiation in adult tissue maintenance and regeneration (Blanc et al., 2016a; Simandi et al., 2015; Zhang et al., 2015). This illustrates the requirement of arginine methylation in the tight regulation of stem cell fate and survival during both embryogenesis and adult homeostasis, which is further supported by the fact that PRMTs can act as reprogramming factors (Nagamatsu et al., 2011; Wang et al., 2016). Arginine Methylation in Diseases The role of arginine methylation in human diseases has been rapidly emerging over the years, especially in cancer. The recent generation of potent and selective inhibitors for type I PRMTs (Eram et al., 2016), PRMT3 (Kaniskan et al., 2015), CARM1 (Ferreira de Freitas et al., 2016), and PRMT5 (Alinari et al., 2015; Chan-Penebre et al., 2015) provides invaluable tools to study arginine methylation in normal cells and cancer. Furthermore, these inhibitors can have therapeutic value in other diseases where PRMTs are dysregulated by overexpression. Cancer PRMTs tend to be upregulated in cancer, and therefore, there are numerous reports of arginine methylation deregulation in cancer. Thus, PRMTs are attractive cancer targets for small molecule inhibition. This subject was reviewed (Greenblatt et al., 2016; Yang and Bedford, 2013); therefore, we will describe the key findings on PRMT inhibitors and recent discoveries. The recent generation of a PRMT5 selective inhibitor (EPZ015666) (Chan-Penebre et al., 2015) offers very promising therapeutic strategies in various cancer types. This orally bioavailable inhibitor demonstrates activity in pre-clinical trials for mantle cell lymphoma, an aggressive form of non-Hodgkin lymphoma disease (Clinical trial: NCT02783300) (Chan-Penebre et al., 2015). Another promising inhibitor is an inhibitor of type I PRMTs (MS023) that crosses the plasma membrane and inhibits aDMA methylation, while increasing MMA and sDMA methylation (Eram et al., 2016), as expected (Dhar et al., 2013). Genome-wide screens to identify genes required for viability of 5-methylthioadenosine phosphorylase (MTAP)-deleted cancer led the identification of PRMT5 and its binding partners, RIOK1, MEP50, and pICIn (Kryukov et al., 2016; Marjon et al., 2016; Mavrakis et al., 2016). Because the physiological role of MTAP is to cleave MTA, these cancers have elevated levels of this metabolite, which is a competitive inhibitor of S-adenosylmethionine. Interestingly, MTA binds the S-adenosylmethionine binding site

of PRMT5, as visualized by crystallography (Mavrakis et al., 2016), and for reasons that remain to be determined, PRMT5 is one of the most sensitive methyltransferases to MTA levels. Thus, MTAP-deleted cells create a vulnerability to PRMT5 depletion by RNA interference (Kryukov et al., 2016; Marjon et al., 2016; Mavrakis et al., 2016). These findings suggest that MTAP-deficient tumors are likely therapeutic targets for PRMT5 deletion. Together, these results show that future research is required to define the role of the catalytic activity of PRMT5 in cancer and the consequences of its inhibition in different cancer types. Leukemia and Lymphoma. PRMT5 expression is upregulated in lymphoma, leukemia, and solid tumors. Myc translocation is known to promote Burkitt’s lymphomas. It was recently discovered that Myc regulates splicing in a PRMT5-dependent manner. MYC upregulates PRMT5 expression, and together they preserve pre-mRNA splicing fidelity that is required to promote cell-cycle progression for cancer cell survival (Koh et al., 2015). Accordingly, depletion of PRMT5 in isolated lymphoma cells subsequently transplanted into nude mice results in tumor-free mice (Koh et al., 2015). PRMT5 activity was shown to promote carcinogenesis by regulating the protein levels of the KLF4 transcription factor. KLF4 methylation by PRMT5 prevents its ubiquitination by VHL, leading to KLF4 accumulation and consequently increased levels of KLF4 targets including breast cancer associated oncogenes. The mutant KLF4R > K was able to disrupt breast tumor initiation and progression, as it leads to KLF4 degradation (Hu et al., 2015). It was reported that inhibition of PRMT5 can also inhibit EBV-driven B cell transformation and thus prevent progression of the malignant phenotype in B cell lymphomas. In mice, overexpressed PRMT5 cooperates with cyclin D1 oncogenic mutant (D1T286A) to promote lymphomagenesis. Cyclin D1T286A accumulates in the nucleus leading to DNA damages and subsequent activation of p53. PRMT5 represses p53 targets via direct methylation of p53, ultimately leading to enhanced tumor cell proliferation without the need for p53 loss-of-function mutations. Importantly, inactivation of PRMT5 reduces oncogenic activity of cyclin D1, c-MYC, NOTCH1, and MLL-AF9, supporting the potential of PRMT5 as a therapeutic target in leukemia/lymphoma (Li et al., 2015b). In normal tissue, PRMT5 is required for hematopoiesis and potentiates both HSC pluripotency and progenitor expansion, suggesting that its inhibition could have myelosuppressive effects (Liu et al., 2015). In AML, PRMT5 has also been reported to promote tumor growth by regulating a feedback loop involving miR-29b, Sp1, and FLT3. Mechanistically, PRMT5 interacts with Sp1, repressing miR-29b expression via H4R3me2s. This leads to an increase of Sp1 levels, usually targeted by miR-29b, which ultimately leads to expression of the receptor tyrosine kinase FLT3, often mutated in AML (Tarighat et al., 2016). Pharmacological inhibition of both PRMT1 and KDM4C impairs the transcription and transformation ability of MLL fusion proteins in acute myeloid leukemia (Cheung et al., 2016). The methylation of the RNA binding protein Aven by PRMT1 regulates the translation of MLL1/4 mRNAs. Thus, depleting PRMT1 halts the growth of T acute lymphoblastic leukemia by preventing protein synthesis of the MLL1 and MLL4 (Thandapani et al., 2015).

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Review Brain Cancer. PRMT5 also emerges as a therapeutic target in glioblastoma, as its expression correlates with tumor grade and progression (Han et al., 2014). Loss of PRMT5 in cancer cells is associated with cell-cycle arrest, p53-independent apoptosis, and reduced migration capability (Yan et al., 2014). PRMT5 is also implicated in neuroblastomas by driving MYCN expression (Park et al., 2015). The absence of PRMT8 in neuronal cells leads to activation of genes involved in gliomagenesis, concurring with the almost complete absence of PRMT8 in human glioblastoma (Simandi et al., 2015). Lung Cancer. PRMT1 and CARM1 are overexpressed in a sample population of 60 patients with non-small cell lung carcinoma (NSCLC) (Elakoum et al., 2014). PRMT1 is involved in NSCLC progression and metastasis via methylation of the transcription factor Twist1, an E-cadherin repressor causing epithelial-to-mesenchymal transition (EMT) (Avasarala et al., 2015). PRMT5 and MEP50 expression is significantly increased in human lung adenocarcinoma and squamous cell carcinomas in comparison to normal tissue. PRMT5 and MEP50 methylate H3R2me2s for gene activation and H4R3me2s for gene repression. Thus, PRMT5 mediates both active and repressive signals on chromatin to support the same biological outcome of increased transcription of genes regulating cell adhesion, morphology, and invasion, all essential for the TGF-b response and cancer metastasis (Chen et al., 2016a). Breast Cancer. CARM1 promotes tumorigenesis and metastasis in breast cancer by methylating R1064 of the chromatin remodeling factor BAF155. This methylation event drives the expression of oncogenes such as those in the c-Myc pathway (Wang et al., 2014a). Recently, it was reported that CARM1mediated methylation of MED12 (RNA polymerase II mediator complex subunit 12), frequently mutated in cancer, sensitizes human breast cancer cells to chemotherapy, as methylation is required for MED12 to repress the expression of the cell cycle inhibitor p21 (Wang et al., 2015). In breast cancer cells, PRMT1 modulates both EMT and cellular senescence through transcriptional activation of Zeb1 by depositing the H4R3me2a activation mark (Gao et al., 2016). PRMT7 expression correlates with decreased survival in breast cancer patients. PRMT7 was shown to promote EMT transition and metastasis by inhibiting E-cadherin expression (Yao et al., 2014) and increasing matrix metallopeptidase 9 expression (Baldwin et al., 2015). Silencing of PRMT7 in MDA-MB-231 breast cancer cells results in the diminution of cell migration and invasion (Baldwin et al., 2015; Yao et al., 2014). Alternative patterns of spliced isoforms of PRMT1, PRMT2, and CARM1 are expressed in breast cancer (Baldwin et al., 2015; Shlensky et al., 2015); however, their roles remain to be defined in this context. Prostate Cancer. PRMT5 was recently demonstrated to contribute to prostate cancer development by functioning as a cofactor for the androgen receptor. The ETS transcription factor ERG is frequently translocated in prostate cancer (TMPRSS2: ERG), and it promotes prostate cancer formation in mice (Afar et al., 2001). PRMT5 was identified as an interactor of ERG and shown to be recruited to androgen receptor target genes for cancer progression (Mounir et al., 2016). Furthermore, it transcriptionally regulates the expression of the androgen receptor

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coding gene by serving as a co-factor for transcription factors Sp1 and Brg1 (Deng et al., 2016). These findings show that PRMT5 is a critical epigenetic regulator promoting the growth of androgen receptor-positive prostate cancer, as it controls androgen receptor gene expression and enhances its tumorigenic activity. Colorectal Cancer. Interestingly, PRMT1 methylates the epidermal growth factor receptor (EGFR) within its extracellular domain in ER/Golgi before its transportation to the cell membrane, enhancing ligand binding and receptor activation (Liao et al., 2015). PRMT1-mediated increased methylation, and consequently over-activation of EGFR signaling, leads to sustained cell proliferation. Methylation-defective EGFR reduced colorectal tumor growth in mice. Importantly, EGFR methylation levels correlated with higher reappearance rates after therapeutic EGFR monoclonal antibody Cetuximab treatment and reduced survival (Liao et al., 2015). Neurodegenerative Diseases Numerous links between arginine methylation and neurodegenerative diseases have been revealed over the last few years. ALS (amyotrophic lateral sclerosis)-linked mutations in the RNAbinding protein FUS (fused in sarcoma) cause it to accumulate in insoluble aggregates in the cytoplasm, and this was alleviated with the depletion of PRMT1 (Tradewell et al., 2012). FUS accumulation is also associated with frontotemporal lobar degeneration (FTLD). MMA harboring FUS was found only in FTLD-FUS, but not in ALS-FUS (Sua´rez-Calvet et al., 2016). The increase of MMA in the absence of PRMT1 concurs with the capacity of the other PRMTs to methylate the hypomethylated substrates (Dhar et al., 2013). Thus, substrate scavenging could be a contributing factor to FTLD-FUS. PRMT5-mediated neuronal death is associated with Alzheimer’s disease. It was proposed that amyloid-beta represses PRMT5 in neurons, leading ultimately to caspase-3-mediated apoptosis (Quan et al., 2015). Mutant huntingtin interacts and co-localizes with PRMT5 in transfected neurons and Huntington disease brains. This interaction is proposed to affect the epigenetic regulation of transcription and RNA processing by PRMT5 (Ratovitski et al., 2015). PRMT6 is involved in polyglutamine diseases such as Huntington disease, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias (Scaramuzzino et al., 2015). PRMT6 has high affinity for a polyglutamine expanded androgen receptor mutant, which is responsible for loss of lower motor neurons and subsequent spinobulbar muscular atrophy (Scaramuzzino et al., 2015). PRMT6 acts as a coactivator, and it can also methylate the androgen receptor. PRMT1 methylation occurs at a RxRxxS/T Akt consensus motif and is mutually exclusive with serine phosphorylation, as is also observed for BAD (BCL2 associated agonist of cell death) (Sakamaki et al., 2011). Arginine methylation at RxRxxS/T motifs are emerging as an important regulation of phosphorylation (Chen et al., 2016b; Scaramuzzino et al., 2015), especially in the context of the nervous system and neurodegenerative diseases, which was recently discussed (Basso and Pennuto, 2015). PRMT5 methylates RNA polymerase II, recruiting the methylarginine-interactor, SMN, to recruit the Sen1 helicase to resolve

Molecular Cell

Review R-loops (Zhao et al., 2016a). Arginine methylation has also been shown to facilitate the resolution of R-loops via TDRD3 and TOP3B, which maintain genomic stability (Yang et al., 2014). As SMN is the affected gene in spinal muscular atrophy, and Sen1 is mutated in ataxia oculomotor apraxia type 2 and ALS, this further emphasizes the link between arginine methylation and neurodegenerative diseases. Although the role of PRMT9 in vivo remains to be determined, PRMT9 was shown to methylate SAP145 at R508, which interacts with the protein SMN, suggesting a role for PRMT9 in neurodegenerative diseases. Interestingly, SMN also interacts with RNA polymerase II once methylated by PRMT5 at R1810 (Zhao et al., 2016a). Reduction of PRMT9 changes the splicing landscape, highlighting a role for PRMT9 in the regulation of alternative splicing machinery in vivo (Hadjikyriacou et al., 2015; Yang et al., 2015). Metabolic Diseases A genetic screen using a cohort of 4,125 families identified PRMT7 as a gene mutated in patients with features of pseudohypoparathyroidism (PHP), also known as Albright hereditary osteodystrophy (Akawi et al., 2015). Although mutations varied from family to family, all PRMT7 mutant carriers were women displaying PHP characteristics including mild intellectual disability, developmental delay characterized by short stature and shortened fourth and fifth metacarpals, rounded faces, and often obesity (Akawi et al., 2015). In mice, PRMTs are also involved in various metabolic processes associated with human disorders. PRMT2 null mice are resistant to dietary-induced obesity (Iwasaki et al., 2010), and PRMT2 is a glucose-sensitive protein (Hussein et al., 2015). PRMT3 was shown to regulate lipogenesis in MEFs, as a cofactor to the liver X receptor a (LXRa), and increased expression of PRMT3 correlates with non-alcoholic fatty liver disease (Kim et al., 2015a). CARM1 was indirectly associated with diabetic retinopathy progression, as it and its associated H3R17me2a mark increased with high glucose, promoting apoptosis of the retinal pigment epithelial cells (Kim et al., 2014a). PRMT5 potentiates hepatic gluconeogenesis in response to fasting by regulating the levels of H3R2me2s on genes involved in gluconeogenesis (Tsai et al., 2013). Since the absence of PRMT5 decreases hepatic glucose production, PRMT5 inhibition in diabetic patients could be useful for lowering blood glucose levels. Aging Arginine methylation is required for maintaining cells in a proliferation state and as such plays a key role in the maintenance of stem cell pools. The loss of PRMTs in transgenic mice has been associated with cellular senescence and premature aging. This has been observed for cells depleted of PRMT1 (Gao et al., 2016), CARM1 (Pang et al., 2013), PRMT5 (Banasavadi-Siddegowda et al., 2016), PRMT6 (Neault et al., 2012), and PRMT7 (Blanc et al., 2016a). In addition, the role of PRMTs in senescence is often associated with aging. The loss of arginine methylation leads to the depletion and exhaustion of stem cells in adulthood. This was observed in various systems including muscle stem cells (Blanc et al., 2016a) and hematopoietic stem cells (Liu et al., 2015). These findings reveal a major epigenetic role for arginine methylation in the aging organism.

Challenges Ahead It is estimated that arginine methylation is as common as phosphorylation and ubiquitination (Larsen et al., 2016). Yet, there are only nine PRMTs while hundreds of kinases and E3 ubiquitin ligases have been identified. Therefore, we can speculate that arginine methylation turnover is not as dynamic as for other modifications, because nine PRMTs must perform the physiological role of hundreds of enzymes. Moreover, not all arginine methylation affects protein function, and thus identifying key functional arginine methylation sites is a constant challenge. The recent identification of dual lysine/arginine demethylases (Walport et al., 2016) raises the question of whether or not these enzymes, which favor lysine in vitro, will impact arginine demethylation in vivo. However, there is a need to identify enzymes that solely or preferentially demethylate methylarginines. Selective PRMT inhibitors will significantly contribute to studying the functional role of arginine methylation. Lastly, defining the entire spectrum of PRMT-specific substrates taking advantage of the PRMTdeficient mice and cell lines is required. In vivo, the simultaneous characterization of multiple hypomethylated substrates is required as the observed phenotype(s) are likely the results of several layers of substrate hypomethylation. These challenges are obviously shared by other fields of post-translational modification, and solving them will continue to help us unravel the mystery of arginine methylation in vivo. ACKNOWLEDGMENTS We thank Javier Di Noia (IRCM) and members of the Richard lab for critically reading the manuscript and for helpful discussions. This work was funded by CIHR MOP-67070 and MOP-93811.

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