*3.2. Arginine Methylation*

Methylation of arginine residues is important for regulating phase separation and recruitment of proteins into MLOs. The side chain of arginine contains a positively charged guanidinium head group that can be multiply methylated. This reaction is catalyzed by protein methyltransferases (PRMTs) using a donor methyl group from S-adenosyl methionine (SAM). Arginine methylation does not change the charge of the side group, but instead alters its volume, charge distribution, hydrophobicity, and potential for hydrogen bonding [53].

Including unmethylated arginine, there are four differential arginine methylation patterns. Arginines can be monomethylated (MMA) or dimethylated (DMA); arginine dimethylation can exist as symmetrical dimethylation (SDMA) or asymmetrical dimethylation (ADMA). Symmetric dimethylation occurs when two methyl groups are added to the two different nitrogen atoms within the guanidino group, whereas asymmetric dimethylation is when two methyl groups are added to the same nitrogen [54]. These reactions are catalyzed by different methyltransferases. PRMTs are grouped into three subtypes based on their catalytic activity. Type I PRMTs include PRMT1, 2, 3, 4, 6, and 8, while type II PRMTs include PRMT5 and 9 and there is one type III methyltransferase, PRMT7 [55,56]. All three classes of PRMTs have the ability to catalyze MMA reactions, but the DMA reactions that occur subsequent to this reaction are specific to type I and II enzymes. Type I PRMT enzymes catalyze the reaction of MMA to ADMA and type II enzymes catalyze MMA to SDMA. PRMT enzymes target arginine residues within glycine-arginine-glycine (GRG) or arginine-glycine-glycine (RGG) sequences [57], which are preferred sites of arginine methylation [58], and are frequently encoded as multiple repeats within low-complexity regions [6].

The hydrogen bond potential of the head group is the same for SDMA and ADMA, but the location of the methyl group can alter the orientation of hydrogen bonding. Additionally, SDMA and ADMA have different electrostatic surface potentials to the head group, resulting in shifting of charge [59]. The hydrophobicity of the head group is also modified upon methylation. Arginine hydrophobicity incrementally increases following the addition of MMA, ADMA, and SDMA, respectively [53]. Hydrophobic residues are generally located within the folded core of proteins, so within the context of an IDR, these changes may have profound effects on the propensity to fold or bind other macromolecules.

The guanidinium electrons are delocalized into π orbitals, enabling interactions via π-stacking [54]. Arginines can form cation-π interactions, which occur between the positively charged guanidinium group and the available electrons of the π orbital of aromatic rings [60]. These cation-π interactions have been shown to drive protein condensate formation [61]. Methylation of arginine has been shown to both favor and disfavor phase separation in certain contexts. ADMA in RGG motifs has been shown to disrupt favorable interactions and thus disrupt phase separation of IDPs such as dead-box helicase 4 (DDX4) [6], heterogeneous nuclear ribonucleoproteins A2 (hnRNPA2) [62], RAP55A [63,64], and FUS [65]. SDMA within RGG motifs, however, has been shown to drive phase separation. In the case of U6 snRNA-associated Sm-like protein (LSM4), SDMA is necessary for phase separation and processing body formation [66]. PRMT5 catalyzes SDMA of LSM4 at multiple arginine residues in its intrinsically disordered C-terminal RGG-containing domain. Mutation of the arginine residues in this domain or knockdown of PRMT5 diminish SDMA and processing body formation in cells [66].

YTH domain-containing family (YTHDF) of proteins contain numerous IDRs, which allows them to undergo LLPS. These proteins are found in cytoplasmic phase-separated SGs, P-bodies, and neuronal RNA granules [67]. In vitro experiments show that enzymatic modifications drive phase separation, but interestingly, the modifications occur to mRNA, not the protein. Methylation of adenosine in RNA, specifically the formation of N6-methyladenosine (m6A), is the most commonly modified nucleotide in mRNA. This modification increases mRNA's multivalency and seeds phase separation of YTHDF proteins in vitro [67]. The protein has the ability to form droplets in solution without the presence of m6A, but only at much higher concentrations. Importantly, the mRNA that seeded droplet formation of YTHDF was multimethylated; unmethylated or singly methylated mRNA did not show droplet formation [67].
