protein arginine methylation: cellular functions and methods of analysis

Biochimica et Biophysica Acta 1764 (2006) 1890–1903www.elsevier.com/locate/bbapap

Review

Protein arginine methylation: Cellular functions and methods of analysis

Steffen Pahlich 1, Rouzanna P. Zakaryan 1, Heinz Gehring ⁎

Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland

Received 7 June 2006; received in revised form 10 August 2006; accepted 21 August 2006Available online 25 August 2006

Abstract

During the last few years, new members of the growing family of protein arginine methyltransferases (PRMTs) have been identified and therole of arginine methylation in manifold cellular processes like signaling, RNA processing, transcription, and subcellular transport has beenextensively investigated. In this review, we describe recent methods and findings that have yielded new insights into the cellular functions ofarginine-methylated proteins, and we evaluate the currently used procedures for the detection and analysis of arginine methylation.© 2006 Elsevier B.V. All rights reserved.

Keywords: Arginine methylation; PRMT; RNA-binding proteins; In vitro and in vivo methylation; Mass spectrometry

1. Introduction

Arginine methylation is a common post-translational modi-fication of mainly nuclear proteins in eukaryotic cells, and iscatalyzed by a family of enzymes called protein argininemethyltransferases (PRMTs). S-Adenosyl-L-methionine (Ado-Met) is used as the methyl donor in this reaction; the methylgroup is being transferred to one of the guanidinium nitrogens ofarginine residues. PRMTs are classified into two groups. Whiletype I PRMTs catalyze the formation ofNG-monomethylarginine(MMA) and asymmetric ω-NG,NG-dimethylarginine (aDMA),type II enzymes form MMA and symmetric ω-NG,N′G-dimethylarginine (sDMA) (Fig. 1A). Although arginine meth-ylation was discovered more than 30 years ago [1], theresponsible enzymes and the wide variety of substrates weremainly described during the last decade. Members of PRMTfamily were found in many different eukaryotes from protozoaand fungi to higher plants and animals as highly conservedenzymes ([2] and references therein;[3]). Currently, nine humanprotein arginine methyltransferases are known: the type Imethyltransferases PRMT1, PRMT3, PRMT4/CARM1,PRMT6, and PRMT8, and the type II enzymes PRMT5,PRMT7, and PRMT9. PRMT2 was identified by sequence

⁎ Corresponding author. Tel.: +41 44 635 5572; fax: +41 44 635 5907.E-mail address: [email protected] (H. Gehring).

1 Both authors contributed equally to this work.

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homology, however, as yet no methyltransferase activity couldbe determined [4] (Table 1). Some homologues from otherspecies are also listed in the table. Crystal structure analysisrevealed a strong structural conservation of the AdoMet-bindingsite of different PRMTs (for reviews, see [5,6]) which howeverdiffer in N- and C-terminal extensions (Fig. 1B). PRMT9structurally differs from all other known PRMTs, implyingconvergent evolution of enzymic mechanism of argininemethylation [7].

PRMT1 is the main methyltransferase in human cells with awide substrate spectrum. It generally recognizes arginineswithin a glycine–arginine-rich (GAR) region, a motif which ispresent in many RNA- or DNA-binding proteins. The other typeI enzymes PRMT3, 6, and 8 also recognize GAR motifswhereas for PRMT4/CARM1 no preferred methylation motif isknown. The type II enzymes PRMT5 and 7 methylate GARmotifs as well as arginines situated not in a known consensusregion. Although the GAR motif is a preferred methylation site,other factors like the 3D structure and the accessibility of thesubstrate are important. PRMT1, for instance, methylates invitro all GAR motifs of the human EWS protein whereasPRMT3 methylates only the C-terminal motif indicating thatstructural determinants prevent or allow specific methylation[8]. Many methylated proteins have been identified to date, butthe responsible methyltransferases are still largely unknown.The known substrates as well as the different subcellularlocalizations of the PRMTs are summarized in Table 1.

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Fig. 1. A: Methylation of the arginine side chain by PRMTs. All PRMTs catalyze the formation of monomethyl-arginine (MMA), where the methyl donor S-adenosyl-L-methionine (AdoMet) is converted to S-adenosyl-L-homocysteine (AdoHcy). In a second step, the type I PRMTs transfer a second methyl group to the sameguanidino nitrogen resulting in an asymmetric dimethylarginine (aDMA) whereas the type II PRMTs catalyze the formation of symmetric dimethylarginines (sDMA).B: Overview of the human PRMT family. The length of each protein is indicated by the numbers of amino acids. The PRMTs 1–8 all have a conserved AdoMetbinding domain (shaded in grey) with the conserved motifs I, post I, II, and III, and a less conserved substrate binding domain (light grey). The conserved motifs I, postI, II, and III are present as well in PRMT 9, but the whole AdoMet binding domain shares only little homology to the other members of the PRMT family.

1891S. Pahlich et al. / Biochimica et Biophysica Acta 1764 (2006) 1890–1903

Subcellular localization of PRMTs indicates the scope ofprocesses these enzymes are involved in. According to somereports, PRMT1 was assumed to localize predominantly in thenucleus [9]. However, this enzyme appears to be highlydynamic with variable subcellular localization and mobilityunder different conditions in various cells. Recently, it wasshown to localize mainly in the cytoplasm, and as a minorfraction in the nucleus [10]. Although PRMT8 is closelyrelated to PRMT1, it is specifically associated with theplasma membrane due to myristoylation of the N-terminal end[11].

2. Arginine methylation and protein function

Many proteins, among them abundant ones such ashistones and RNA-binding proteins, have been shown tocontain methylarginines (for a review see [2]). Most of theknown methylated proteins are substrates of PRMT1, whichis the predominant type I PRMT in mammalian cells,accounting for 85% of cellular PRMT activity [12]. Argininemethylation is implicated in many cellular processes including

transcription, RNA processing and transport, translation,signal transduction, DNA repair, apoptosis etc. Argininemethylation increases bulkiness of the affected residue and itshydrophobicity, blocks hydrogen bonding, but, in contrast tophosphorylation or acetylation, does not alter its charge. Themaintenance of charge is particularly important becausepositively charged arginines often participate in bindingother proteins or nucleic acids. Methylation most likely hasa less pronounced effect on interactions mediated by arginineresidues. It seems that arginine methylation has a mildereffect on proteins than other posttranslational modifications,modulating certain processes rather than acting as an on/offswitch.

There is accumulating evidence that some substrates aremethylated by both type I and type II PRMTs (the pre-mRNA-binding protein SmB by CARM1 or PRMT5, histone 4 byPRMT1 or PRMT5). Thus, aDMA- and sDMA-methylation ofthe same proteins may regulate distinct protein–proteininteractions, leading to opposing biological consequences, andfurther expanding the functional diversity of the proteome ([13]and references therein, Table 1).

Table 1Intracellular localization of the PRMTs and their known substrates

PRMT, intracellular localization Substrate Ref.

PRMT1 (type I),nuclear and cytoplasmic

53BP1 [58]CIRP [109]EBNA-1 [110]EWS [8]FGF-2 [111]Fibrillarin [112]FKBP 12 [113]Fmrp [44]GRP33 [31]GRY-RBP [85]GST-GAR [87]Hepatitis C virus NS3 Helicase [52]Hepatitis delta antigen S-HDAg [45]Histone H4 [1]hnRNP A1 [114]hnRNP A2 [35]hnRNP K [85]hnRNP R [85]ILF3 [115]Mre 11 [116]NF 90 [115]NIP45 [117]Nucleolin [118]p137GP1 [85]PABP II [43]PGC-1α [24]QKI-5 [31]RBP58 [85]RNA helicase A [30]SAF-A (hnRNP U) [119]Sam68 [120]SAMT1 [85]SLM-1 [31]SLM-2 [31]SPT5 [20]TAFII68 [85]TIS21/BTG2 [70]TLS/FUS [85]ZF5 [85]

HMT1 (yeast homolog)nuclear and cytoplasmic

Gar1p [103]Hrp1 [121]Nab2p [34]Nor1p [103]Npl3 [122,123]Nsr1p [103]Yra1 [26]


not determined [124]

HRMT1L1, (yeast homolog)nuclear

Adenovirus hnRNP E1B-AP5 [125]

PRMT3 (type I),cytoplasmic

EWS [8]GST-GAR [87]hnRNP A1 [114]PABP II [43]rpS2 [46]Sam68 [120]

PRMT4/CARM1 (type I),nuclear

CARM1 [18]Histone H3 [126,127]HuD [27]HuR [28]ILF3 [42]

Table 1 (continued)

PRMT, intracellular localization Substrate Ref.

p300/CBP [18]PABP1 [42]TARPP [128]

DART4 (Drosophila homolog),nuclear

Squid [129]Vasa [129]

PRMT5 (type II),nuclear and cytoplasmic

coilin [91]EBNA-1 [110]EBNA-2 [130]Histones H4, H2A, H3 [17,131]LSm4 [132]MBP [94]SM B/B′ [133]Sm D1 [132]Sm D2 [132]SPT5 [20]

PRMT6 (type I),nuclear

DNA polymerase beta [59]GST-GAR [9]HIV Tat [21]HMGA1a [22]Np13 [9]PRMT6 [9]


GST-GAR [134]

PRMT8 (type I),membrane-associated

GST-GAR [11]

PRMT9 (type II),nuclear and cytoplasmic

not determined [7]

Unknown PRMT Golgi proteins [135]Herpes simplex virus ICP27 [136]Myosin [137]RBP16 [138]Vav-1 [48,49]

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2.1. Transcriptional regulation

2.1.1. Histone arginine methylationMethylation of nucleosomal histones by PRMTs plays an

important and dynamic role in nuclear receptor-mediatedtranscriptional regulation, chromatin remodeling and otheraspects of gene expression (reviewed in [5,14]). Linkage ofhistone arginine methylation to other types of histonemodifications, such as lysine methylation, acetylation, phos-phorylation and ubiquitination, regulates binding of specificinteracting proteins, altering chromatin structure and transcrip-tion process [15]. Three PRMTs have been reported to catalyzehistone methylation: CARM1 methylates Arg 2, Arg 17 andArg 26 of histone H3; PRMT1 methylates Arg 3 of histone H4;and PRMT5 methylates Arg 8 of histone H3 and Arg 3 ofhistone H4 (Table 1). PRMTs may be targeted to specific genesand to broader regions of euchromatin and heterochromatin. Inmammals, formation of aDMA in histones by PRMT1 andCARM1 participates in gene activation while formation ofsDMA by PRMT5 is associated with gene repression (reviewed


in [16,17]) and conversion of histone methylarginine residuesto citrulline by peptidylarginine deiminase 4 (PAD4) leads totranscriptional repression. The cellular events following histonearginine methylation remain poorly understood; recent findingsindicate that protein arginine methylation, in general, isrequired for mammalian development and important forcellular proliferation and differentiation (for reviews, see[5,14]).

2.1.2. Arginine methylation of nonhistone proteins involved intranscription

Arginine methylation participates in the regulation of theinitiation and elongation steps of transcription. PRMTs havebeen shown to methylate, in addition to histones, the histoneacetyltransferase CREB binding protein (CBP), which func-tions with the related p300 protein as a key transcriptionalcoactivator. The KIX domain of CBP has been shown to bemethylated by CARM1 in vitro, thereby blocking CREBactivation. Thus, CARM1 functions as a corepressor in cAMPsignaling pathway via its methyltransferase activity while actingas a coactivator for nuclear hormone receptors [18]. CARM1specifically methylates conserved arginine residues in vitro andin vivo in another domain of CBP [19]. Together, these dataprovide strong evidence that arginine methylation represents animportant mechanism for modulating co-activator transcrip-tional activity. PRMT1 and PRMT5 have been shown tomethylate the transcriptional elongation factor SPT5 and thus toregulate its interaction with RNA polymerase II [20]. Argininemethylation by PRMT6 inhibits the transactivation activity ofHIV Tat protein, therefore PRMT6 acts as a restriction factor forHIV replication [21]. The high-mobility group protein A1(HMGA1a), a modulator of chromatin structure and transcrip-tion has been demonstrated to be specifically methylated byPRMT6 in vitro [22] and in vivo at Arg 57 and Arg 59, whichare embedded in the region critical for both protein–DNA andprotein–protein interactions [23]. Methylation of peroxisomeproliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) by PRMT1 leads to the induction of genes important formitochondrial biogenesis [24].

2.2. RNA processing and export; protein translation

RNA-binding proteins (RBPs) participate in processing,folding, stabilization and localization of RNAs and mRNAtranslation. Most of RBPs possess GAR motifs, and are well-known or putative substrates for PRMTs. Arginine residues inthe binding sites of RBPs have been recognized as key aminoacids in RNA–protein interactions. The guanidinium nitro-gens of arginine residues favor ionic interactions andhydrogen bonding. Due to methylation, the affinity of aparticular RBP for its RNA targets decrease, if the methylgroup prevents hydrogen bonding by steric hindrance.Alternatively, arginine methylation may positively influenceRNA–protein interactions, as the arginine residue becomesmore hydrophobic due to addition of methyl groups and mayengage in more van der Waal interactions. The consequencesmay be facilitated stacking with the bases of the RNA ([13]

and references therein). In the following, the experimentalevidence for both promotion and inhibition of protein–RNAinteraction is discussed. Arginine methylation has beenreported to facilitate the packaging of ribonucleoproteincomplex [25,26], and to synchronize transcription withmRNA processing. Methylation of the RNA-binding proteinsHuD [27] and HuR [28] by CARM1 has been shown toregulate their function in stabilization of specific mRNAswhich is important in cell proliferation and differentiation.Several RBPs lack proper subcellular localization in theirhypomethylated state [29,30]. Deletion of the methylationsites or use of methylation inhibitors caused accumulation ofSam68 in the cytoplasm and prevented Sam68-mediatedexport of HIV RNAs [31]. Inhibition of methylation results ina significant decrease in nuclear accumulation of highmolecular weight fibroblast growth factor 2 [32]. In yeast,the PRMT1 homolog Hmt1/Rmt1 methylates the hnRNPsNp13p, Hrp1p [33], and Nab2p [34], implying a role ofmethylation in facilitating mRNA export from the nucleus.Particularly, methylation of mRNA export factor Npl3 leadsto its dissociation from the transcriptional elongation factorTho2, a process important for the subsequent export of themRNA [26]. Arginine methylation promotes the nuclearlocalization of hnRNP A2 [35]. Recently, it was reported thatthe arginine methylation sites in the RGG2 and RGG3 boxes,assist the nuclear localization of EWS protein [36]. However,replacement of arginine residues in RGG boxes by lysines didnot disturb the transcriptional activity of EWS protein [37],indicating that the EWS protein is imported into the nucleuseven in the absence of arginine residues. This finding isconsistent with the results demonstrating that the absence ofRGG boxes of EWS protein as well as inhibition of argininemethylation by adenosine dialdehyde (AdOx) did not affectthe subcellular distribution of EWS protein [38]. Recently, anunknown nuclear localization signal (C-NLS), which isresponsible for the nuclear import and retention of the EWSprotein, has been identified [39].

PRMT5 is found in at least three different protein complexes,two nuclear and a cytoplasmic one, in which it performs themethylation of Sm proteins and coilin, which is an importantstep in snRNP biogenesis [40,41].

The poly(A)-binding proteins I and II are methylated byCARM1 [42] and PRMT1 [43], respectively, however, thefunctional effect of these methylations is not known as yet.Arginine methylation has been identified as an additionalmechanism for modifying the function of fragile X mentalretardation protein (Fmrp) and was suggested to limit ormodulate RNA binding of Fmrp [44]. PRMT1 methylates asmall form of the hepatitis delta antigen (S-HDAg) in vitroand in vivo, which is essential for HDV RNA replication,especially for replication of the antigenomic RNA strand toform the genomic RNA strand. Methylation-defective HDAglooses the ability to form a speckled structure in the nucleusand even permeates into the cytoplasm [45]. The methyl-ation of ribosomal protein S2 is conserved from yeast toman and has been shown to influence ribosomal biosynthe-sis [46,47].


2.3. Signal transduction

Cell signaling is controlled by posttranslational modifica-tions that alter protein–protein interactions. More and more datashow that arginine methylation is used to mediate signaltransduction downstream of the TCR (T-cell antigen receptor),cytokine (including interferon), and NGF receptors (forreviews, see [2,13]). The TCR signaling pathway controlledby CD28 has been shown to promote arginine methylation ofthe nucleotide exchange factor Vav1 [48,49]. A role of proteinarginine methylation in regulating the expression of interleukin2 in T lymphocytes has been reported. The recruitment ofmethylarginine-specific protein(s) to cytokine promoter regionswas shown to regulate the cytokine gene expression [50]. TheJAK-STAT pathway downstream of cytokine receptors has beenreported to be regulated through arginine methylation ofSTAT1, STAT3 and STAT6 transcription factors [51–53].However, arginine methylation on STAT1 and STAT3 remainsquestionable, as the methylation could not be confirmed in cellculture or in vitro [54,55].

2.4. DNA repair

Facts arise that arginine methylation plays a significantrole in signaling DNA damage. It was demonstrated that theGAR motif of MRE11 is methylated by PRMT1. Methylationof MRE11 did not affect the complex formation of theMRE11–RAD50–NBS1 double-strand break-repair protein,however, substitution of arginine residues in the GAR motifinhibited the exonuclease activity of MRE11 by regulating itsassociation with nuclear structures, such as the PML nuclearbodies and the sites of DNA damage ([56] and referencestherein). Another central mediator of the DNA damagecheckpoint, 53BP1, was shown to be methylated. It is rapidlyrecruited to sites of DNA double-strand breaks and formscharacteristic nuclear foci, demonstrating its role in the earlyevents of detection, signaling and repair of damaged DNA.Whereas oligomerization of 53BP1 was shown to beindependent of methylation [57], asymmetric dimethylationof the GAR motif of 53BP1 by PRMT1 was demonstrated tobe required for DNA binding activity [58]. Whether themethylation of the arginine residues in the GAR region isnecessary for DNA binding, is not defined yet. PRMT6 wasfound to play a direct role in the regulation of DNA repairprocesses by methylation of DNA polymerase β, whichparticipates in base excision repair [59].

2.5. Protein–protein interactions

Arginine methylation can increase or decrease protein–protein interactions. The methylation of an arginine residue ofthe HIV-1 Nef protein blocks the interaction with tyrosinekinase Fyn. The substrate of the Src kinase in mitosis, Sam68, isan adaptor protein that binds to several proteins containing SH3domain. The methylation of arginines neighboring prolinemotifs in Sam68 actually prevents association with SH3domains. The interaction between the nuclear factor of activated

T cells (NF-AT) and the NF-AT-interacting protein of 45 kDa,NIP45, decreased in the presence of methylase inhibitors ([2]and references therein). Methylation of arginine residues byPRMT1 in hnRNP K did not influence RNA-binding activity,the translation inhibitory function and the cellular localizationof the protein, but reduced the interaction of hnRNP K with thetyrosine kinase c-Src leading to inhibition of c-Src activationand hnRNP K phosphorylation [60].

There is evidence that methylated arginines serve asphysiological ligands for the Tudor domain. The Tudordomains of the splicing factor SPF30 and the Tudor domain-containing protein TDRD3 interact with GAR motifs in amethylarginine-dependent manner, and with methylarginine-containing cellular proteins [61]. The Tudor domain of 53BP1can act as an adaptor mediating intramolecular as well asintermolecular protein–protein and protein–nucleic acidassociations [62]. Arginine methylation of RBPs, Sm B, B′,D1, and D3 by PRMT5 mediates their assembly into maturespliceosomal small nuclear ribonucleoprotein particles(snRNPs) due to the binding to the Tudor domain of thesurvival of motor neuron (SMN) protein. Consistent withthese data, pre-mRNA splicing reactions were inhibited, andSm proteins accumulated in the cytoplasm when argininemethylation was inhibited [61]. Contrary, a symmetricallydimethylated RG-peptide derived from the EWS proteindecreased its interaction with the Tudor domain of SMN [63].Taking into account that native EWS protein interacting withSMN in vivo [63] is excessively asymmetrically dimethylatedby PRMT1 [8,64], one might hypothesize that aDMA ofEWS protein may stimulate its binding to SMN, whereassDMA formation impairs this interaction.

2.6. Arginine protection

In addition to the above mentioned functions, it wasproposed that arginine methylation might protect crucialarginine residues against attack by endogenous reactivedicarbonyl agents, such as methylglyoxal and other naturalby-products of normal metabolic pathways [65]. The condi-tions, if these highly cytotoxic compounds are not detoxifiedand removed, are associated with inactivation of proteins andare linked to diabetes, cancer, neurodegenerative diseases andpathophysiologies of aging.

2.7. Regulation of PRMT activity

The enzymic activity of PRMTs can be modulated byregulation of their expression, by formation of the PRMTmacromolecules, by changing accessibility of the enzyme to thesubstrate, as well as by other posttranslational modifications,and protein–protein interactions.

2.7.1. Homo-oligomerization of PRMTsThe assumption that protein arginine methylation is

participating in signaling indicates that it should be regulated.It has been reported that PRMT6 and CARM1 displayautomethylation activity, although the effects are not yet


described. Formation of homo-dimers or larger homo-oli-gomers has also been linked to the activity of Hmt1, PRMT1,and PRMT5 ([5] and references therein). Enzymic activity ofPRMT1 or Hmt1/Rmt1 is abolished when dimerization ofthese enzymes is prevented [66,67]. However, later it wasreported that PRMT1 and PRMT5 are catalytically activeonly in the form of multimers, but not as a dimer or tetramerof the expressed subunit. It was proposed that some uncha-racterized additional factor(s) may multimerize PRMTs invivo [68] and most probably affect PRMT activity.

2.7.2. Regulation of arginine methylation by otherposttranslational modifications

Recent studies have suggested a cooperation betweendifferent histone modifications during transcription. It wasreported that acetylation of the histone H3 at Lys 18 and Lys 23tethers recombinant CARM1 to the H3 tail and inducesmethylation of Arg 17 [69]. PRMT5 can methylate hypoace-tylated H3 and H4 more efficiently than the hyperacetylatedforms. Particularly, acetylation of Lys 9 prevents the methyl-ation of Arg 8 in H3 [17]. Taking into account these and otherresults, Bedford and Richard suggested that preexistingmodifications close to a site of methylation can alter therecognition motif of protein methyltransferases. A similar eventwas proposed with HIV Tat, where the known lysine acetylationsites are next to the PRMT6 methylation motif ([13] andreference therein).

2.7.3. Regulation of PRMT activity by PRMT-binding proteinsPRMTs bind not only their substrates to be methylated, but

also several other proteins and protein complexes, which canaffect the methyltransferase activity of the PRMTs under certainconditions. The related proteins BTG1 and TIS21/BTG2 bindPRMT1 and stimulate its activity toward selected substrates[70]. Binding of the tumor suppressor DAL-1 to PRMT3 acts asan inhibitor of enzymic activity, both in in vitro reactions and incells [71]. DAL-1 also regulates PRMT5 activity by eitherinhibiting or enhancing it in a substrate-specific manner [72].CARM1 is found in a complex of at least ten proteins called thenucleosomal methylation activator complex (NUMAC) [73].CARM1 within NUMAC is able to methylate nucleosomalhistone H3, whereas recombinant CARM1 preferentiallymethylates free histone H3. Nuclear PRMT5 forms a complexwith the hSWI/SNF chromatin remodelers BRG and BRM, andthis association enhances PRMT5 methyltransferase activity[17]. Protein phosphatase 2A (PP2A) inhibits PRMT1 enzymicactivity and therefore not only increases the helicase activity ofNS3 (a hepatitis C virus protein, which is inhibited bymethylation) but also interferes with the cellular defense againstviruses by inhibiting interferon-induced signaling throughSTAT1 [74].

2.8. Arginine methylation as a source of free MMA, aDMA andsDMA

Proteolysis of methylated proteins is the cellular source offree mono- and dimethylarginine residues. MMA and aDMA

are known to be endogenous competitive inhibitors of nitricoxide synthase (NOS), which synthesizes NO from L-arginine,and plays an important role in cardiovascular system.Dimethylarginine dimethylaminohydrolases (DDAHs) specifi-cally hydrolyze MMA and aDMA, but not sDMA. Deregulatedlevels of these free methylarginines affects NOS activity and,consequently, may result in cardiovascular disease ([75] andreferences therein).

2.9. Demodification of methylated arginines

Over the years, arginine methylation has been consideredto be an irreversible posttranslational modification. The searchfor arginine demethylases did not yield any results.Nevertheless, the fact that lysine demethylases such asLSD1 have been identified makes it possible that argininedemethylases may also exist. Two groups showed thatarginine methylation of histones can be reduced in vitro bydeimination with PAD4 [76,77]. This reaction converts bothunmodified arginine and monomethylarginine, but notdimethylarginine, to citrulline, an amino acid that is nottranscriptionally incorporated into proteins. Modeling studiessuggest that asymmetrically and symmetrically dimethylatedarginine cannot be accommodated in the active site of PADbecause of steric occlusion [78]. Although PADs canindirectly antagonize arginine methylation, they are not realdemethylases because citrulline lacks the positively chargedguanidino group and, thus, cannot functionally substitute forarginine. Therefore, the conversion of histone monomethy-lated arginines to citrulline by PAD4 does not represent thesecond half of an on/off switch. To accomplish this cycle,each citrulline residue would have to be converted back toarginine in situ. Enzymes that convert peptidyl citrulline toarginine, as described with the amino acid citrulline areunknown leaving up to now only the scenario of the removalof citrulline by degrading citrullinated proteins. Nevertheless,deimination of monomethylated arginines seems not to be themain mechanism of regulation of this posttranslationalmodification. Whereas the PRMTs are present in allinvestigated cell types, PADs are specifically expressed inhighly differentiated cells of skin (PAD1), skeletal muscle(PAD2), hair follicles (PAD3) and mature granulocytes(PAD4). Thus, in most cells it is impossible to demodifymethylated arginine residues with the known PADs. Inaddition, most of methylated proteins contain aDMA andsDMA (as an example, 30 aDMAs are found in EWSprotein), which cannot be converted to citrulline by PADs,leaving the question whether citrullination has a specific roleother than just removal of mono-methylated arginines.

3. Methods to detect and identify arginine methylatedproteins and their methylation sites

3.1. In vitro methylation with [3H]-AdoMet

A common way to analyze arginine methylation is the invitro methylation by using recombinant methyltransferases,


radioactively labeled S-adenosyl-L-[methyl-3H]-methionine([3H]-AdoMet), and purified proteins or cell extracts assubstrate. The labeled methyl groups are transferred to thesubstrates and can be detected by fluorography [79]. However,in eukaryotic cells most methyl-accepting sites are occupiedindicating that methylation occurs soon after translation [80,81]and untreated cell extracts are not suitable as substrate. Toobtain free methylation sites of methyl accepting proteins,cellular protein methyltransferases are usually inhibited by 5′-methyl-thioadenosine (MTA) [82] or by adenosine dialdehyde(AdOx). AdOx inhibits S-adenosyl-L-homocysteine hydrolase,leading to an accumulation of intracellular S-adenosyl-L-homocysteine, which in turn is a product inhibitor of themethyltransferases [83]. Cells treated with AdOx or MTAaccumulate newly synthesized unmethylated methyl-acceptingproteins during the incubation period [51,81] and can be used assubstrate for in vitro methylations. However, one has toconsider that the expression level of originally methylatedproteins might be affected by the inhibitor treatment [38]. Trisbuffer is not recommended for in vitro methylation becausesome PRMTs are inhibited in its presence [84]. Due to theincorporation of the labeled methyl group, methylation activityof the used enzyme can be detected by fluorography afterseparation of the proteins by SDS-PAGE. Using hypomethy-lated cell extracts, different substrates as well as substratespecificities of the PRMTs were observed with this method[9,84–87]. It is easy and rapid to detect or proof methyl-transferase activity of PRMTs against single substrate proteinsor hypomethylated cell extracts. However, when hypomethy-lated cell extract is used as substrate, an identification ofunknown methylated proteins is difficult and no informationabout the position of the methylated arginines within theproteins can be gained. For identifying the site of themethylation, arginines may be substituted by site-directedmutagenesis, but this is time consuming. Determining thenumber of incorporated methyl groups per protein is often

Fig. 2. Detection of protein arginine methylation by peptide mass fingerprints. PotenHere, recombinant (unmethylated) human EWS protein was in-gel digested and analprior methylated with PRMT1. Methylation leads to a mass shift of multiples of 1technique can also be used to identify methylation sites of endogenously methylated pto be compared with theoretical digests of the (unmethylated) protein to identify thehas to be confirmed by MSMS peptide sequencing (see Fig. 3) because adducts fro

inaccurate with this method, as absolute quantification of theincorporated radioactivity is difficult.

3.2. Identification of the type of methylation by amino acidanalysis

The type of methylation, namely MMA, aDMA, or sDMA,can be distinguished by amino acid analysis of the methylatedsample. The methylated proteins or peptides have to behydrolyzed completely and the resulting amino acids have tobe separated chromatographically [70,88]. MMA, aDMA, andsDMA differ in their retention times or Rf-values and can beassigned with the corresponding standards.

3.3. Detection of methylated arginines by antibodies

The different types of arginine methylation can also beanalyzed by Western blotting using methyl-arginine-specificantibodies. This technique was used to identify the asymmetricdimethylation of the histones H3 and H4 [89,90], or SAM68[31], and the symmetric dimethylation of coilin [91]. Theconversion of unmodified or monomethylated arginines in H3and H4 to citrulline by PAD4 were also visualized by usingantibodies against citrulline and monomethylated arginine[76,77].

Commercially available antibodies (SYM10, SYM11,ASYM24 from Upstate or 7E6 from Abcam) are derived fromsynthetic peptides of several RG-repeats including either sDMAor aDMA [91] or as in the case of histones from a peptidecovering the exact sequence including the modified arginine[76]. These antibodies do not necessarily recognize all and onlymethylated proteins. With the 7E6-antibody, cross-reactivitiescould not be excluded [81], and also the specific recognition ofmethylated STAT1 and STAT3 by antibodies is questionable[55]. So for each analysis, the specificity of the used antibodieshas to be tested.

tial methylation sites of a protein can be identified by peptide mass fingerprint.yzed by MALDI-TOF MS. The same was done with recombinant EWS protein,4 Da of peptides containing potential methylation sites like RGG motifs. Theroteins, which are purified from a cell extract. The resulting peptide masses havemass shifts. Once, potential methylated peptides are identified, the modificationm sample preparation can also lead to a mass shift of 14 Da (see Section 3.4).

Fig. 3. MSMS spectrum of a peptide containing asymmetrically dimethylated arginines. A methylated peptide of the human EWS protein was sequenced by MALDI-TOFTOF-MS to determine and confirm the methylation sites. Methylated peptides are typically rich in arginines and glycines yielding in few fragments with lowintensities. However, the presence of asymmetrically dimethylated arginines (dmR) can be confirmed by the appearance of the ion atm/z 46 (dimethylamine, dma) andits loss from the parent ion ([M+H]+ −45). The most intense signals in this spectrum (y4 and b7) result from the preferred C-terminal fragmentation after an asparticacid.


3.4. Detection of methylated arginines by mass spectrometry(MS)

MS has become a favorable method not only to identifyproteins by peptide mass fingerprint, but also to characterizepost-translational modifications of proteins.

Methylation leads to a mass increase of 14 Da perincorporatedmethyl group at the arginine residue. After enzymicdigestion of a methylated protein, the mass shift can be used tofind peptides that potentially contain this modification (Fig. 2).However, some artifacts can arise during sample preparationwhich also results in a mass increase of 14 Da. Methanol-basedprotein staining procedures, for instance, give rise to theformation of methyl-esters at the acidic side chains of glutamic-

Fig. 4. In vitro methylation of a synthetic peptide. A synthetic peptide (human nuclepoints, aliquots of the reaction were analyzed by MALDI-TOF MS. The incorporatiomethylation of the peptide is complete as indicated by the single signal of the eight folto determine enzymic activities, KM's, or methylation properties of PRMTs (e.g. seq

or aspartic acid. Also cysteine residues alkylated withacrylamide and carbamidomethylated cysteines differ in massby 14 Da. Since the above mentioned adducts occur frequentlyduring sample preparation, the arginine methylation has to beverified by tandem mass spectrometry (MSMS) (Fig. 3). MSMSanalysis will also identify the modified amino acid(s) in peptidescontaining several potential methylation sites.

MS can be used either to detect the methylation sites ofendogenously methylated proteins purified from a cell extract[64,92] or to monitor the in vitro methylation of recombinantunmodified proteins with different PRMTs [8,93]. A furtheradvantage of MS is its sensitivity. Amounts in the pmol down tofmol range are sufficient and from our experience it is moresensitive than the alternative method (Section 3.1). However,

olin residues 676–692) was methylated with PRMT1. After three different timen of each methyl group result in a mass shift of 14 Da. At time point three, thed methylated peptide in the spectrum. This method can be used for kinetic studiesuence preferences).


the entire protein sequence may not be covered completely bythe detected peptides. Especially tryptic digests of proteinscontaining GAR motifs, the preferred methylation sites, canresult in small peptides that are below the detection limit of amass spectrometer and therefore, potential methylation sitesmight be missed. Previously, it was reported, that trypsin doesnot cleave after dimethylated arginines [94,95]. However, in arecent study using MSMS-analysis, the occurrence of suchcleavages was demonstrated [8], so a missed cleavage site is notnecessarily a proof for a methylated arginine. To increasesequence coverage, the analysis should be performed with atleast two proteases of different cleavage specificities.

Enzymic activity of recombinant PRMTs can be measuredwith MS by in vitro methylation of defined peptidescontaining potential methylation motifs (e.g. a syntheticpeptide corresponding to residues 676–692 of humannucleolin has been proven to be a very useful substrate)(Fig. 4). Each incorporated methyl group can be detected dueto the mass shift and followed over the time. Besidesdetermining the initial velocity of the enzymes, informationabout the sequence and sites of the methylation reaction canbe obtained by analyzing intermediate products. Thereby,information about the sequence preferences and the substratespecificity of the PRMTs can be gained [8].

Modifications like protein phosphorylation introduceadditional negative charges to the modified peptide resultingin a bad ionization behavior for mass spectrometry. Suchpeptides have to be enriched or modified prior to MS analysisto get sufficient signals. Arginine methylation, however, doesnot alter the charge of the arginine side chain and theionization efficiency of a modified and unmodified peptide iscomparable during MALDI-TOF MS. Even quantitativeanalyses of the methylation reaction can be performed bycalculating the peak ratios of an unmodified and modifiedpeptide in a spectrum yielding the kinetic parameters of theinvestigated enzyme [8].

Precursor Ion Scanning is frequently used for the detectionof several post-translational modifications such as phosphor-ylation on Ser, Thr, or Tyr residues [96,97], O- and N-linkedglycosylation [98], or acetylation [99]. Collision induced decayof peptides results in the typical sequence-specific y-type andb-type ions, and non-sequence specific ions like internalfragment ions or immonium ions (Fig. 3). Fragmentation ofmodified peptides by MSMS leads also to fragment ionsunique for each modification, like the diagnostic ion PO3

− at m/z 79 (in the negative mode) for phosphopeptides. In a precursorion scan the full mass range is scanned, but only peptidesgenerating these modification-specific reporter ions areselected and further analyzed, resulting in a spectrum contain-ing only modified peptides. There are two fragment ionsappearing exclusively due to the fragmentation of a dimethy-lated arginine residue which are suitable for a precursor ionscan. One fragment ion at m/z 71.061 corresponds todimethylcarbodiimidium, which can be generated from thefragmentation of both, aDMA and sDMA-containing peptides.Thus, a discrimination of the two isoforms is not possible. Asecond specific fragment ion at m/z 46.06 corresponding to

dimethylammonium is exclusively generated from the frag-mentation of aDMA-containing peptides and can be used as aspecific reporter ion for the detection of aDMA (Fig. 3). It wasshown that precursor ion scanning of m/z 46.06 using trypticdigests of aDMA containing proteins or mixtures of unmod-ified peptides with aDMA containing peptides in varying ratiosresulted in the assignment of all aDMA containing peptides ofthe samples [100,101].

4. Proteome analysis of arginine methylated proteins

The methods described above are mainly suitable for thecharacterization or identification of single proteins and theirmodifications. For a proteomic approach, however, the wholeproteome of an organism or cell type needs to be analyzed. Toreduce sample complexity, “subproteomes” like nuclear,cytoplasmic, and membrane proteomes or only phosphorylatedor glycosylated proteins (phospho- or glycoproteome) arecurrently used. The proteins of interest are enriched by methodslike differential centrifugation, immunoprecipitation of proteincomplexes or by affinity chromatography selective for modifiedresidues.

Enrichment and analysis of a “methyl-proteome”, however,is difficult because the incorporation of methyl groups to thearginine side chain changes the physicochemical properties ofthis residue only slightly. Although the hydrophobicity isenhanced, the charge remains unchanged and methylatedproteins or peptides cannot be separated, so far, fromunmodified ones by any chromatographic method as e.g.phosphopeptide enrichment by immobilized metal affinitychromatography (IMAC).

Methylated proteins are dominantly RNA- or DNA-bindingproteins and located mainly in the nucleus, so a nuclear extractcontains an elevated level of methylated proteins compared to awhole cell extract. However, cytoplasmic proteins or proteinsattached to the membrane were also found to be methylated andwould be missed in such an analysis. In the following, methodsare described to enrich or visualize methylated proteins for a“methyl-proteome” analysis.

4.1. In vivo methylation with [3H]-AdoMet

Yeast cells are capable to take up the methyl donor AdoMetfrom the medium [102]. Proteins from yeast cells, grown in[3H]-AdoMet-containing medium were separated by 1D or 2Dgel electrophoresis and the methylated proteins were detectedby fluorography [103]. AdoMet-based methylation of lysines,histidines, or nucleic acids leads also to radioactive labeling. Inrat cells, however, it has been shown that almost 90% ofmethylated amino acids of the cell extract are mono- andasymmetrically dimethylated arginines [104]. If one considers asimilar proportion of methylated amino acids in yeast,methylation of other residues than arginines is expected to bea minor problem. The in vivo labeling, however, is limited to theanalysis of protein methylation in yeast, because most othereukaryotic cells have only a poor uptake efficiency of AdoMet[105].


Instead, Najbauer et al. [104] used a hypomethylated cellextract of PC12 rat cells to visualize methyl acceptor proteins.PC12 cells were incubated in culture prior to lysis with AdOx,and [3H]-AdoMet was then added to the cell extract tomethylate the proteins by endogenous methyltransferasespresent in the extract. The proteins were separated by 2D gelelectrophoresis and more than 50 different methyl acceptorproteins, mainly basic proteins, could be detected byfluorography. However, an identification of these proteinswas not possible and methylation of hypomethylated cellextracts might not reflect the “methyl-proteome” of normallygrown cells.

To avoid hypomethylation, cells can be incubated inmethionine-free medium containing labeled methionine in thepresence of protein synthesis inhibitors like chloramphenicoland cycloheximide. The labeled methionine is taken up by thecells and converted intracellularly into the labeled methyl-donorAdoMet. The incorporation of the labeled methyl group to aprotein is due to the AdoMet-based methylation process and notdue to incorporation of labeled methionine into newlysynthesized proteins. With this method, Liu et al. [106]discovered the methylation of numerous heterogeneous nuclearribonucleoproteins (hnRNPs) by immunopurification and 2Dgel electrophoresis of hnRNP complexes and detection of thelabeled methylated hnRNPs by fluorography.

4.2. Immunopurification of methylated proteins

Boisvert et al. [107] used antibodies derived againstsynthetic peptides containing either sDMA or aDMA indifferent RG-rich motifs (see above) to immunoprecipitatemethylated proteins out of a HeLa cell extract. The precipitatedprotein complexes were digested and more than 200 proteinswere identified by LC/MSMS sequencing of the peptides.Proteins were assumed to have methylated arginines if theycontain GAR-motifs within their amino acid sequence but wereabsent in a control experiment. Peptides containing methylatedarginine residues, however, were not directly identified.Discrimination between potential asymmetric or symmetricdimethylated proteins was based on the used antibodies anddiscrimination between methylated and co-purified unmethy-lated proteins was not possible.

Ong et al. [108] introduced heavy methyl SILAC (stableisotope labeling by amino acids in cell culture) to identifymethylated proteins from cell extracts. Cells were grown inmedium containing either “light” (CH3)- or “heavy” (13CD3)-methionine, which is taken up by the cells and metabolicallyconverted to labeled AdoMet, used for intracellular methylationevents. Both samples were combined in a 1:1 ratio, subjected toa single immunopurification step with specific antibodies (seeabove), digested, and analyzed by high mass accuracy LC/MSMS. The incorporation of one “heavy”methyl group leads toa mass increase of 4 Da compared to the “light” one. Thus, ionpairs with equal intensity ratios and distances corresponding tothe incorporation of one or multiple methyl groups (e.g. 4 Da forMMA, 8 Da for DMA) are hints for methylation. Peptides withsuch ion pairs were sequenced by MSMS to identify the

position of the methylated arginine residues. With thispromising method, both, identification and relative quantifica-tion of the methylation site are feasible. However, to reducesample complexity, an immunopurification step with antibodiesagainst methylated arginines was performed, and only 33methylated proteins out of a whole HeLa cell extract wereidentified in this study. This modest enrichment of methylatedproteins again reflects the moderate specificity of currentlyavailable antibodies against methylarginines for immunopre-cipitation of the “methyl-proteome”.

In summary, a variety of methods for the analysis of proteinarginine methylation are currently available, especially for invitro studies. A complete “methyl-proteome” analysis canreveal the number of methylated proteins in the cell and give anidea of the variety of cellular processes this modification isinvolved in, but this approach remains a challenge. Highthroughput mass spectrometry makes it possible to identifyhuge amounts of different proteins and peptides in a short time,but there is still a lack of methods in efficiently enrichingmethylated proteins out of a whole proteome.

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protein arginine methylation: cellular functions and methods of analysis

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