Arginine methylation is a prevalent post-translational modification found on both nuclear and cytoplasmic proteins. The methylation of arginine residues is catalyzed by the protein arginine N-methyltransferase (PRMT) family of enzymes. Proteins that are arginine methylated are involved in a number of different cellular processes, including transcriptional regulation, RNA metabolism and DNA damage repair (Bedford and Richard, 2005). Most PRMTs methylate glycine- and arginine-rich patches (GAR motifs) within their substrates. The complexity of the methylarginine mark is enhanced by the ability of this residue to be methylated in three different ways on the guanidino group: monomethylated (MMA), symmetrically dimethylated (sDMA) and asymmetrically dimethylated (aDMA), each of which has potentially different functional consequences.
Mammalian arginine methyltransferases
There are three structurally defined types of S-adenosylmethionine (AdoMet)-dependent methyltransferase (Katz et al., 2003). The largest class (Class I) has a common seven-stranded β-sheet structure. The Class II enzymes are the SET lysine methyltransferases and Class III encompasses the membrane-associated methyltransferases. PRMT family members fall into Class I and harbor a set of four conserved sequence motifs (I, post-I, II, and III) and a THW loop (Katz et al., 2003). Motifs I, post-I and the THW loop form part of the AdoMet-binding pocket (Zhang et al., 2000). Ten mammalian PRMTs have been identified to date. Eight have been shown to catalyze the transfer of a methyl group from AdoMet to a guanidino nitrogen of arginine, generating S-adenosylhomocysteine (AdoHcy) and methylarginine. No activity has yet been demonstrated for PRMT2 and PRMT9.
PRMTs are classified as type I, type II, type III or type IV enzymes. Types I, II and III enzymes methylate the terminal (or ω) guanidino nitrogen atoms. Type I and type II enzymes all catalyze the formation of an MMA intermediate, then type I PRMTs (PRMT1, 3, 4, 6 and 8) further catalyze the production of aDMA, whereas type II PRMTs (PRMT5, PRMT7 and FBXO11) catalyze the formation of sDMA. PRMT7 also exhibits type III enzymatic activity – the propensity to catalyze the formation of MMA on certain substrates and not proceed with sDMA catalysis. A type IV enzyme that catalyzes the monomethylation of the internal (or δ) guanidino nitrogen atom has been described in yeast.
PRMT1, the predominant mammalian type I enzyme, was identified by sequence similarity to the yeast arginine methyltransferase Hmt1/Rmt1 (Lin et al., 1996). PRMT1 is broadly expressed and localizes to both the cytoplasm and the nucleus and has substrates in both these cellular compartments (Herrmann et al., 2005). PRMT1 methylates a number of hnRNP molecules, and this modification plays a role in the shuttling of these proteins between the cytoplasm and the nucleus (Herrmann et al., 2004). PRMT1 also methylates histone H4 at arginine 3 (Wang et al., 2001), thus contributing to the histone code. This modification on histone H4 functions as a transcriptional activation mark, which could either result in the recruitment of methyl-binding proteins or influence the deposition of other posttranslational marks in the vicinity. As a transcriptional coactivator, PRMT1 is recruited to promoters by a number of different transcription factors (Bedford and Richard, 2005). The central role that PRMT1 plays as a regulator of protein function is revealed by the disruption of this enzyme in mice. PRMT1-knockout mice die shortly after implantation (Pawlak et al., 2000). The crystal structure of PRMT1 in complex with the reaction product (AdoHcy) and a GAR motif has been described (Zhang and Cheng, 2003).
The first relative of PRMT1 to be identified was PRMT2 (Katsanis et al., 1997). A novel feature of PRMT2 is that it harbors an SH3 domain at its N-terminus (Scott et al., 1998). Although PRMT2 does not have enzymatic activity, it does function as a coactivator for the estrogen receptor (Qi et al., 2002). PRMT2-null mice are viable and grossly normal (Yoshimoto et al., 2006).
A unique property of PRMT3 is that it harbors a zinc-finger domain at its N-terminus, which is its substrate-recognition module (Tang et al., 1998). The 40S ribosomal protein S2 (rpS2) is a zinc-finger-dependent substrate of mammalian PRMT3 (Swiercz et al., 2005). Importantly, in fission yeast this same enzyme-substrate pair (PRMT3-rpS2) exists (Bachand and Silver, 2004), and the disruption of the prmt3 gene in this organism results in an imbalance in the 40S:60S free subunit ratio. Mouse embryos with a targeted disruption of PRMT3 are small, but survive after birth and attain a normal size in adulthood. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, which demonstrates that it is an in vivo PRMT3 substrate (Swiercz et al., 2007).
CARM1, sometimes referred to as PRMT4, was identified in a yeast two-hybrid for proteins that associate with GRIP1, the p160 steroid receptor coactivator (Chen et al., 1999). The recruitment of CARM1 to promoters results in the methylation of histone H3 at Arg17 and of other coactivators including p300/CBP and AIB1 (Bedford and Richard, 2005). CARM1-mediated methylation has a positive effect on transcription. CARM1 is not only a steroid receptor coactivator but also enhances transcription/translation rates in pathways responding to other transcription factors (Bedford and Richard, 2005). In addition, CARM1 methylates splicing factors and regulates the coupling of transcription and splicing (Cheng et al., 2007). CARM1-null mice die just after birth and are smaller than their wild-type littermates (Yadav et al., 2003). Cells from CARM1-null embryos have defective estrogen receptor and NF-κB pathways. CARM1 has also been implicated in the epigenetic programming of early embryos (Torres-Padilla et al., 2007). Finally, the fact that CARM1 is a coactivator for nuclear receptors makes it a likely candidate for over-expression in prostate and breast cancers. Indeed, increased expression of CARM1 correlates with androgen independence in human prostate carcinoma (Hong et al., 2004) and CARM1 is overexpressed in breast tumors (El Messaoudi et al., 2006).
PRMT5 was cloned as Jak2-binding protein and shown to methylate histones H2A, H3 and H4 (Branscombe et al., 2001; Pollack et al., 1999). It localizes to both the cytoplasm and the nucleus. In the cytoplasm, PRMT5 is found in the `methylosome', where it is involved in the methylation of Sm proteins, which implicates it in snRNP biogenesis (Friesen et al., 2001). Nuclear PRMT5 associates with regulators of transcriptional elongation SPT4 and SPT5 (Kwak et al., 2003). Nuclear PRMT5 also forms complexes with the hSWI/SNF chromatin-remodeling proteins BRG and BRM, where it is responsible for methylating Arg8 on histone H3 (Pal et al., 2004) and is required for muscle differentiation (Dacwag et al., 2007). PRMT5 and H3R8 methylation levels are elevated in lymphoid cancer cells (Pal et al., 2007). A general note of caution should be added here: the Reinberg group have found that αFLAG M2-agarose enriches for PRMT5 activity (Nishioka and Reinberg, 2003); thus many affinity-purified FLAG-tagged complexes are `contaminated' with PRMT5.
PRMT6 is restricted to the nucleus and it has the ability to methylate itself (Frankel et al., 2002). Like PRMT1, PRMT6 methylates a GAR motif. However, it displays unique substrate specificity – it methylates histones H3 and H4 in vitro, whereas PRMT1 only methylates histone H4 (Lee et al., 2004). DNA polymerase β was found to form a complex with PRMT6. Methylation of Pol β by PRMT6 strongly stimulates DNA polymerase activity (El-Andaloussi et al., 2006). Thus, PRMT6 plays a role regulating DNA base excision repair. Finally, PRMT6 has also been shown to methylate a number of HIV proteins (Invernizzi et al., 2007).
PRMT7 was first identified in a genetic screen for susceptibility to chemotherapeutic cytotoxicity (Gros et al., 2003). It is one of two PRMTs that harbor two putative AdoMet-binding motifs (Miranda et al., 2004). It has a strong propensity to catalyze the formation of MMA but not DMA on a fibrillarin-derived peptide substrate (Miranda et al., 2004). Miranda et al. thus classified PRMT7 as a type III enzyme. Using a different peptide substrate, Lee et al. showed that PRMT7 catalyzes the formation of sDMA, consequently classifying it as a type II enzyme (Lee et al., 2005b). It is possible that distinct substrates are methylated in different fashions by this enzyme. A study that focused on identifying loci that impart susceptibility to drug-induced nephropathy has implicated PRMT7 as a candidate (Zheng et al., 2005). Also, PRMT7 plays a role in male germline imprinted gene methylation through its interaction with CTCFL (a protein that associates with the imprinting control region) and subsequent methylation of histone 4 Arg3 (Jelinic et al., 2006).
PRMT8 was identified through its high degree of sequence identity to PRMT1 (Lee et al., 2005a). PRMT8 has a unique N-terminus that harbors a myristoylation motif that facilitates its association with the plasma membrane. It is largely restricted to the brain.
FBXO11, also referred to as PRMT9 (2p16.3), was identified as a potential PRMT because it has regions that display weak sequence similarity to the I, post-I, II and III amino acid sequence motifs (Cook et al., 2006). Unlike other PRMTs, it does not harbor a THW loop; however, FLAG-tagged hFBXO11 has been reported to have type II activity (Cook et al., 2006), but HA-tagged hFBXO11 and its C. elegans ortholog (DRE-1) have been reported not to have PRMT activity (Fielenbach et al., 2007).
Proteins that harbor GAR motifs are often targets for PRMTs, although CARM1 is an exception and cannot methylate a GAR motif. Recent advances in mass spectrometry mean that more and more isolated methylation sites that are not within GAR motifs are being identified. In addition, both CARM1 and PRMT5 can also methylate PGM motifs (Cheng et al., 2007). PGM motifs are proline-, glycine-, methionine-, arginine-rich patches that are found in a number of splicing factors (Bedford et al., 1998). PRMT substrates have been extensively reviewed recently (McBride, 2006; Pahlich et al., 2006).
Methylation regulates protein-protein interactions
Arginine methylation facilitates the interaction of GAR and PGM motifs with Tudor domains. The symmetric dimethylation of SmB by PRMT5 is required for its interaction with the Tudor domains of SMN, SPF30 and TDRD3 (Cote and Richard, 2005). The asymmetric dimethylation of CA150 by CARM1 also provides a docking site for the Tudor domain of SMN (Cheng et al., 2007). Thus, motifs harboring either aDMA or sDMA residues bind a subset of Tudor-domain-containing proteins. It is likely that a conserved aromatic `cage' in Tudor domains is the methyl-binding pocket (Sprangers et al., 2003).
Arginine methylation can also act as a negative regulator of protein-protein interactions. For example, the methylation of arginine residues adjacent to a proline-rich motif can block binding to SH3, but not WW, domains (Bedford et al., 2000). A second example is the CARM1-mediated modification of the GRIP1-binding domain of p300 (Lee et al., 2005c). Finally, histone H3 methylation at Lys4 provides a docking site for the double chromodomains of CHD1 (chromo-helicase/ATPase DNA-binding protein 1). The histone H3 Arg2 site is reported to be methylated by CARM1, and this methylation together with Lys4 methylation (H3K4me3R2me2a) decreases the binding affinity fourfold relative to histone H3 Lys4 methylation alone (Flanagan et al., 2005).
Regulation of arginine methylation
PRMT-binding proteins can regulate the activity of PRMTs. They can inhibit, activate, or change the substrate specificity of PRMTs. The related proteins BTG1 and TIS2/BTG2 bind to PRMT1 and stimulate its activity towards selected substrates (Lin et al., 1996). The BTG1-binding protein hCAF1 also regulates PRMT1 activity (Robin-Lespinasse et al., 2007). Binding of the tumor suppressor DAL-1 to PRMT3 acts as an inhibitor of enzyme activity, both in vitro and in cell lines (Singh et al., 2004). CARM1 is found in a complex of at least 10 proteins called the nucleosomal methylation activator complex (NUMAC) (Xu et al., 2004). CARM1 within NUMAC acquires the ability to methylate nucleosomal histone H3, whereas recombinant CARM1 preferentially methylates free histone H3. PRMT5 forms complexes with the hSWI/SNF chromatin remodelers BRG and BRM, and this association enhances PRMT5 methyltransferase activity (Pal et al., 2004). The binding of CTCFL to PRMT7 has also been reported to elevate the activity of this PRMT (Jelinic et al., 2006).
Arginine residues within proteins can be converted to citrulline by deimination. A major group of deiminated proteins are the core histones H2A, H3 and H4 (Nakashima et al., 2002). The peptidyl arginine deiminases (PADs) can block methylation on an arginine residue by converting it to citrulline (Cuthbert et al., 2004; Wang et al., 2004). PADs catalyze the deimination of arginine, but not MMA or DMA, to citrulline (Raijmakers et al., 2007). Thus, peptidyl arginine deiminases are not demethylases. However, these enzymes may carry out a preemptive strike on key sites of arginine methylation, thereby preventing subsequent methylation. The first evidence that PRMTs themselves are regulated by posttranslational events was recently described (Higashimoto et al., 2007). In this case, the phosphorylation of CARM1 results in a decrease in PRMT activity. There are no reported arginine demethylases and this topic is an active area of research.
Note added in Proof
Thank you to Steven Clarke for his comments on this review. M.T.B. is supported by NIH grant DK62248.