RG Motif Regions in RNA Binding and Phase Separation

Review KDC

YJMBI-65769; No. of pages: 16; 4C:

RGG/RG Motif Regions in RNA Binding and Phase Separation

P. Andrew Chong 1 , Robert M. Vernon 1 and Julie D. Forman-Kay 1, 2 1 - Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON, Canada 2 - Department of Biochemistry, University of Toronto, Toronto, ON, Canada

Correspondence to Julie D. Forman-Kay: Program in Molecular Medicine, Peter Gilgan Centre for Research and Learning, 686 Bay St., Toronto, ON, Canada M5G 0A4. [email protected] https://doi.org/10.1016/j.jmb.2018.06.014 Edited by Richard W. Kriwacki

Abstract RGG/RG motifs are RNA binding segments found in many proteins that can partition into membraneless organelles. They occur in the context of low-complexity disordered regions and often in multiple copies. Although short RGG/RG-containing regions can sometimes form high-affinity interactions with RNA structures, multiple RGG/RG repeats are generally required for high-affinity binding, suggestive of the dynamic, multivalent interactions that are thought to underlie phase separation in formation of cellular membraneless organelles. Arginine can interact with nucleotide bases via hydrogen bonding and π-stacking; thus, nucleotide conformers that provide access to the bases provide enhanced opportunities for RGG interactions. Methylation of RGG/RG regions, which is accomplished by protein arginine methyltransferase enzymes, occurs to different degrees in different cell types and may regulate the behavior of proteins containing these regions. © 2018 Elsevier Ltd. All rights reserved.

Introduction RGG/RG repeats were first identified in the mid1980s, with papers on the adenovirus hexon-assembly protein [1, 2], nucleolin [3] and fibrillarin [4]. Involvement of RGG/RG repeat-containing proteins in phase separation processes has sparked renewed interest in these regions. Protein phase separation refers to the emergence of a protein-enriched phase and a proteindepleted phase from a uniformly dispersed solution [5–8]. The enriched phase often assumes the form of liquid droplets containing highly concentrated protein, akin to oil droplets in an oil–water mixture. Protein phase separation is now understood to underlie the formation of membraneless organelles and enables cells to compartmentalize specific molecules and functions, much like membranes do. Unlike membrane-enclosed organelles, formation and dissolution of membraneless organelles is readily controlled by post-translational modifications [9–11], protein concentration [12] or environmental cues [13], providing a powerful mechanism for cellular regulation. Examples of membraneless organelles include the nucleolus, P-bodies and stress granules, with roles that include, but are not limited to, 0022-2836/© 2018 Elsevier Ltd. All rights reserved.

ribosome biogenesis [14], mRNA translational repression and degradation [15] and stalling of mRNA translation, respectively [15]. These membraneless organelles are frequently termed ribonucleoprotein (RNP) bodies or granules, because they generally contain both proteins and RNA. This review will provide a molecular-level perspective on RGG/RG motif interactions with RNA, their involvement in phase separation in vitro and within cellular RNP bodies/membraneless organelles and regulation of their interactions by methylation. Since RGG/RG motifs were first described, they have been identified in many other proteins, often occurring as multiple repeats. In humans, there are greater than 100 proteins with at least two RGG motifs that are spaced less than 5 residues apart and greater than 1700 proteins with at least two RG motifs that are spaced less than 5 residues apart [16]. As shown in Table 1, there is a large range in the number of RGG/ RG repeats found in proteins. The FET proteins [fused in sarcoma (FUS), Ewing's sarcoma protein (EWS) and TATA binding associated factor 15 (TAF15)] contain approximately 20 RGG repeats each, while FMR1 contains only 2 full RGG repeats. This range in J Mol Biol (2018) xx, xxx-xxx

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

Table 1. Selection of RGG/RG repeat-containing proteins Proteina

RG

RGGb

Caprin1 CHTOP Poly-GR (associated with C9orf72 repeat expansions)

11 23 Variable

3 9 0

Ddx4 EWS Fibrillarin FMR1 FUS

10 30 16 9 24

4 22 9 2 19

FXR1 FXR2 GAR1 G3BP1 hnRNPA1 hnRNPA2/B1 hnRNPU KHDR1 Kmt2b Lsm14a nucleolin SERBP1 TAF15

9 8 19 7 9 7 19 14 35 17 12 15 31

2 0 21 2 4 4 7 2 6 10 10 7 22

Ubap2l Hrp1p Saccharomyces cerevisiae Nab2p S. cerevisiae Npl3p S. cerevisiae Scd6 S. cerevisiae Laf-1 Caenorhabditis elegans PGL-1 C. elegans PGL-3 C. elegans cabeza Drosophila melanogaster Cirp2 Xenopus laevis

3 6 4 19 9 8 12 12 12 8

14 3 4 15 1 19 10 6 9 4

Other repeatsb Short polyglutamine Repeat-associated non-ATG translation leads to expression of GR (or RG) polymers in addition to GA, GP, PA, PG and PR polymers SGG × 2, PGG × 8, N-terminal Q,G,S,Y,T,A,P-rich region Polyglycine YGG × 1, SGG × 9, multiple polyglycine, N-terminal Q,G,S,Y, T-rich region Short polyarginine Short polyproline, polyarginine, polyserine Polyglycine YGG × 5, SGG × 2, FGG × 6, polyglycine Polyglycine Polyglycine, polyglutamate Polyproline Polyglutamate, polyglycine, polyproline, polyglutamine FGG × 4, polyglutamine, polyaspartate Polyglycine, polyalanine YGG × 22, SGG × 10, YGGDRGG × 12, N-terminal Q,G,S, Y-rich region Short polylysine, polyserine and polythreonine Polyglutamine Polyalanine and polyglycine repeats YGG × 6 Many polyglycine repeats

a

Proteins are human unless indicated otherwise. Note that RGG is a subset of RG. The documented repeats are not independent. For example, the YGG and RGG in YGGDRGG are accounted for twice in this table. b

the number of RGG repeats can have an effect on the affinity/avidity of interactions formed by these RGG/RG regions [17, 18], as well as on multivalency, key for phase separation [19]. RGG/RG repeats usually occur in low-complexity regions, intrinsically disordered regions (IDRs) composed largely of limited amino acid types, or as part of intrinsically disordered proteins (IDPs) [20]. These IDRs and IDPs do not assume a single folded structure but instead rapidly interconvert between highly heterogeneous conformational states. IDRs that phase separate are usually low complexity [21]. RGG/RG repeats are sometimes associated with other repetitive sequences, including polyalanine, polyaspartate, polyglutamate, polyglutamine and polyglycine repeats, as well as other diglycine-containing motifs such as FGG, PGG, SGG and YGG (Table 1). These accompanying features, which vary between RGG regions, likely modulate their interactions, although the effect of these features on RGGcontaining regions is not well studied. Some associated sequence features, such as FG repeats [22] and polyglutamine and polyglutamine/asparagine-rich

repeats [23–26], are also know to promote phase separation and/or aggregation on their own. Note that GR and GGR sequences are also quite common in RGG/RG-containing proteins, leading to the term glycine–arginine-rich (GAR) regions, which specifies content but not sequence.

RGG Interaction with RNA RGG-containing regions are RNA-binding regions. This was first realized when the RGG/RG region of hnRNPU was shown to be responsible for the RNA binding properties of the protein [27]. The authors also pointed out that the RGG region was likely “unordered, extended and flexible,” which was at odds with the prevailing paradigm in the early 1990s that proteins needed to assume a folded structure to function. Binding was demonstrated for RNA homopolymers, including poly(G), poly(U), poly(A) and to much lesser extent poly(C). Interaction with single-stranded DNA was found to be less efficient than binding to RNA [27]. Although RNA homopolymer binding has not been

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

explored for most RGG/RG repeat-containing proteins, there are other reported examples, including EWS, which is able to bind to poly(G) and poly(U) RNA via its C-terminal RGG region [28]. Weaker binding to singlestranded DNA versus RNA was also noted in this case [28]. Interaction of RGG motifs with poly(ADP-ribose), or PAR, a polymer composed of two or more ADPribose nucleotides covalently joined at the ribose group, has also been reported [29]. RGG Interaction with G-quadruplex Structures Many RGG:RNA interactions are expected to be highly dynamic interactions, described in more detail below. As a result, there is very little direct structural information about these interactions. The structural information that we do have comes from the interactions between fragile X mental retardation protein (FMRP) and a high-affinity artificial ligand, which was identified by screening and which can form a Gquadruplex [30]. Although we suspect that Gquadruplex structure recognition is not a general feature of RGG/RG motif ligands [31], here we describe some of the data on G-quadruplexes in detail because of the structural data and the large body of literature on these interactions. To gain insight into RGG/RG recognition of RNA and identify RNA molecules that bind to FMRP, Darnell et al. [30] performed a screen using 96-mer RNA molecules with 52 random bases in the middle. The result of this screen was a consensus recognition motif containing four di-guanosine repeats, suggesting that the RGG/RG region in FMRP recognizes an RNA G-quadruplex structure, a hypothesis supported

by mutations. A large-scale microarray study of FMRP-interacting mRNAs indicated that two-thirds of them contain sequences capable of forming Gquadruplexes, substantiating G-quadruplexes as a target for RGG/RG motifs [32]. G-quadruplexes are known to be stabilized by potassium and destabilized by lithium, a fact that was used to confirm that FMRP binds to the G-quadruplex structure [33]. Further support was provided by structures of the high-affinity synthetic G-quadruplex-containing ligand identified by the Darnell screen [30], called sc1, in complex with a peptide corresponding to the RGG region of FMRP [34, 35]. The first structure confirmed that the sc1 RNA folds into a G-quadruplex structure and that a peptide from FMRP containing the sequence RGGGGR binds at the junction between the duplex and quadruplex regions (Fig. 1). Notably, binding could be disrupted by mutating any of the residues in the RGGGGR, which is consistent with the well-defined structure observed for this segment. Significantly, the sc1 Gquadruplex is suspected to be more stable than many natural G-quadruplexes [35–37]. Several reasons lead us to question the generality of RGG/RG motif recognition of G-quadruplex structures. First, RGG regions can bind to RNA homopolymers that do not form G-quadruplexes [e.g., poly(U) and poly(A)]. Second, the exact RGGGGR sequence required for high-affinity binding is rare and is not found in FUS, EWS, TAF15, nucleolin, hnRNPU, Ras GTPase-activating protein-binding protein 1 (G3BP1), SERPINE1 mRNA-binding protein 1 (SERBP1) or Lsm14a. Third, rather than binding to the Gquadruplex itself, FMRP binds at the junction of the duplex and quadruplex and the nucleotide sequences

Fig. 1. Structure of an FMRP RGGGGR peptide bound to the sc1 RNA. (a) The duplex region of the sc1 RNA is colored in purple. The quadruplex region is colored in yellow, with orange highlighting for the guanines that form the three guanine quadruplexes. The FMRP peptide is colored in cyan, with the arginine residues highlighted in blue. (b) An extracted guanine quadruplex plane rotated by 90° to show the quadruplex structure (PDB: 5de5). Hydrogen bonds are shown as black dashed lines.

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

that make up these junctions vary. Fourth, Gquadruplex structures can vary considerably and they can be formed by one [34], two [38], three [39] or four [40] strands, comprising RNA or DNA [41]; thus, there is no common structure to be recognized by RGG repeats. Fifth, experiments using lithium, which is known to destabilize the sc1 G-quadruplex, demonstrate that the G-quadruplex is not required for binding to some RGG/RG motifs (e.g., FXR2P) [33]. Collectively, these indicate that the G-quadruplex is not generally required for recognition by RGG/RG motifs. G-quadruplexes are also not as uniformly stable as suggested by the sc1 RNA [37]. To our knowledge, the FMRP:sc1 complex is the only RNA:RGG complex for which a structure has been determined and the interaction may be a special case of a highaffinity relatively rigid interaction formed by a short RGG-containing region. The ability of an oligonucleotide to fold into a single G-quadruplex conformer depends on its length [42, 43]. The sc1 RNA itself appears to sample multiple conformations in the absence of the FMRP peptide leading to broadening of NMR spectra. G-quadruplexes have also been shown to be substantially destabilized in mammalian cells [44]. Although the mechanism that melts or inhibits formation of G-quadruplexes in cells is not defined, cellular phase-separated protein bodies may destabilize G-quadruplexes, in the same way that condensed Ddx4 melts nucleic acid duplexes [45] (see below). Destabilization of G-quadruplexes by RGG-containing peptides has been demonstrated in vitro [36, 46], which suggests that RGG peptides may bind to unfolded forms of the oligonucleotides. Together these data support the idea that G-quadruplex RNA recognition is not a general feature of RGG regions and that sequences with the potential to form G-quadruplex structures are less stable in the cell than suggested by the FMRP:sc1 structure. Critical Role of Arginines in Oligonucleotide Binding Analysis of structures of protein:DNA and protein: RNA complexes indicates that arginines are an important element of nucleic acid binding segments of proteins [47]. The positively charged guanidino group enables an electrostatic interaction with and can hydrogen bond with the negatively charged phosphate backbone (Fig. 2a). The guanidino group can also form favorable π-stacking, cation–π (Fig. 2b) and hydrogen bond interactions with the ribose sugar (Fig. 2c) and bases (Fig. 2d). Arginine is involved in one-third of all of the hydrogen bonds between amino acids and DNA observed in a set of 129 protein:DNA complex structures from the Protein Data Bank (PDB) [48]. Interestingly, the same study indicates that two-thirds of arginine:base hydrogen bonds are with guanine. In contrast, arginine:cytosine hydrogen bonds make up only 5% of these arginine:base hydrogen bonds, probably because cytidine base hydrogen bonds are

already satisfied in a double-stranded DNA helix. There are far fewer structures of proteins bound to RNA, making it more difficult to collect statistics. Nevertheless, arginine plays a similarly important role in RNA binding, involved in one quarter of all hydrogen bonds with RNA [49]. The ratio of hydrogen bonds with nucleotide bases over hydrogen bonds with the phosphate backbone is higher in RNA:protein interactions compared to DNA:protein interactions, as might be expected from the greater number of unpaired bases in RNA [49]. Roughly 58% of protein contacts with RNA and ssDNA are made with nucleotide bases compared to 24% of protein contacts with dsDNA [49]. Arginine side chains are also the largest contributor to RNA:protein π–π planar stacking interactions [21]. The ability to be simultaneously involved in hydrogen bonding, π-stacking and cation–π interactions may explain an advantage for binding to DNA/RNA structures that are not in double-helix structures. For example, at the transition of the duplex to quadruplex region of the sc1 G-quadruplex, arginine is able to simultaneously stack between and hydrogen bond with nucleotide bases (Fig. 3a). This hints that any RNA structures that allow for arginine to π stack and hydrogen bond with bases may be high probability targets for RGG/RG motifs. Tyrosine in the known RNA-binding motif YGG [50] is also able to stack and hydrogen bond with nucleotide bases, providing further support for this aspect of the interaction. Thus, the role of arginines in RNA and DNA binding is well established and access to nucleotide bases increases possible interactions. Snapshots of RG Motif Interactions with RNA As indicated, there are few examples of structures of RGG regions bound to RNA, probably because many of the individual RGG interactions with RNA are dynamic and low-affinity. Recent NMR data on the interaction of the third FUS RGG-rich region and Gquadruplex-containing DNA and RNA oligonucleotides support the idea that the RGG region remains dynamic in the complex [42, 51]. However, we can get snapshots of these transient interactions by looking for RG dipeptides in high-resolution crystal structures of various folded proteins binding to RNA. Such structural snapshots demonstrate a variety of interaction possibilities, including π–π stacking, cation–π, hydrogen bonding, charge–charge/dipole, van der Waals and hydrophobic. Specifically, arginine can hydrogen bond with the phosphate backbone (Fig. 3a, c), with guanine and adenine bases (Fig. 3a, d) and with the ribose 2′hydroxyl (Fig. 3b). Π-stacking or cation–π interactions are also observed with cytosine, guanine, adenine and uridine bases (Fig. 3a–d). The glycines in RGG/RG motifs likely confer the conformational flexibility to form local structural elements important for RNA binding and contribute via π-stacking with the peptide bond [21, 34, 35]. Hydrogen bonding between the glycine

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

Fig. 2. Structures illustrating modes of arginine binding to RNA with adjacent simplified schematic diagrams. Atoms in structures are colored by atom type, with oxygen in red, phosphate in orange, nitrogen in blue and protein carbons and RNA carbons shown in green and cyan, respectively. Rods are used to illustrate planar π–π stacking interactions. Rods are placed normal to the planar interaction, extend to a carbon VDW radius of 1.7 Å and are colored by category with amino acid side chain groups in purple and RNA in gray. Hydrogen bonds are shown with yellow dashed lines. In the schematics, the RNA is shown in blue, while the arginine guanidino is shown in green. (a) Arginine hydrogen bonding with three different phosphate groups (PDB: 2zko). (b) Arginine forming π-stacking/cation–π arrangement with an adenine base (PDB: 5kla). (c) Arginine hydrogen bonding to ribose hydroxyl groups (PDB: 4lgt). (d) Arginine hydrogen bonding with the guanine base (PDB: 4lgt).

carbonyl and amide groups and the nucleotide bases (Fig. 3b) and the ribose 2′-hydroxyl (Fig. 3c) is also observed. The variety of interactions with RNA observed in these examples of RG sequences binding to RNA/DNA suggests a mechanism through which RGG/RG regions can bind to a wide range of RNA sequences. In agreement, one study of the RGG/RGcontaining protein PGL-3 suggests that it can bind most mRNA sequences [52]. Oligonucleotide Structure and Protein Binding Oligonucleotide secondary structure elements may be important for RGG/RG repeat binding in the sense that structures that expose the nucleotide bases for interaction with arginine and glycine may be more easily bound. This may explain the preference of the RGG/RG repeat-containing protein Ddx4 for singlestranded DNA or RNA over double-stranded DNA or RNA [45, 53], since base stacking and many of the hydrogen-bonding sites are satisfied in the double helix. Single-stranded RNA and DNA were found to partition into a Ddx4 protein-rich droplet independent

of oligonucleotide length. In contrast, only short double-stranded DNA oligonucleotides could partition into the Ddx4 droplets. Fluorescence resonance energy transfer studies provided evidence that these short double-stranded DNA molecules were melted within the droplets, presumably because intermolecular protein interactions with the nucleotide bases were more favorable than the intramolecular base stacking and hydrogen bonding within these short oligonucleotides [45]. Likewise, experiments done with ribosomal RNA (rRNA) indicated that heat denaturation of the rRNA was required to promote PGL-3 droplets, probably because melting opens up the RNA for interaction with RGG/RG repeats in PGL-3 [52]. However, it may not be structure or lack thereof per se that is important, but rather enhanced access to the nucleotide bases, either in bulges and stem loops [54] or in single-stranded regions. This is consistent with the RGG interaction with the quadruplex–duplex junction, where the bases are accessible. Thus, the data suggest that RGG/RG motifs are more likely to bind to RNA conformations that expose their bases for recognition.

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

Fig. 3. Examples of arginine:RNA interactions in folded protein structures. Color coding as in Fig. 2, with the addition of hydrogens shown in white for the NMR structure in panel a. (a) R15 of the FMRP peptide (PDB: 2la5) hydrogen bonds with the G′ 7 nucleotide base (′ is used to distinguish nucleotides from amino acids), forms a salt bridge with the phosphate of G′6 and π-stacks between the A′17 and C′5 nucleotide bases. Residues are numbered as in the PDB file. The structure of the displayed residues is very similar in the NMR (PDB: 2la5) and crystal (PDB: 5de5) structures. (b) Arginine π-stacking with cytosine and guanine bases and hydrogen bonding with the 2′ hydroxyl of ribose. Hydrogen bonds between the adjacent glycine residue and the cytosine base are also observed (PDB: 3r2c, 1.9-Å resolution). (c) Arginine guanidino group π-stacking with a cytosine base and hydrogen bonding/salt bridge with the phosphate backbone. The adjacent glycine hydrogen bonds with the ribose sugar of the cytidine (PDB: 4r3i, 1.8-Å resolution). (d) Arginine stacking in place of a nucleotide base in an RNA helix, with hydrogen bonding to the opposing base, π-stacking with the adjacent bases, and hydrogen bonding of the arginine backbone amide and the nucleotide phosphate (PDB:4lgt, 1.3-Å resolution).

RGG Phase Separation RGG/RG Motifs in Cellular RNA Granules Many RGG/RG repeat-containing proteins are localized to micron-sized membraneless organelles that are visible in micrographs of cells [5–8]. Ddx4 is an important component of perinuclear granules or nuage in mammals and Drosophila [55, 56], analogous to Caenorhabditis elegans P-granules [55] that contain Laf-1, PGL-1 and PGL-3 [57]. Nucleolin and fibrillarin are two important components of the nucleolus [4, 58, 59]. Stress granules contain members of the FET family—FUS, EWS and TAF15, as well as FMRP, G3BP1 and caprin-1 [60–66]. Lsm14a

(also known as RAP55) is required for P-body formation and is also found in stress granules [11, 67, 68]. All of these proteins contain RGG/RG regions. Notably, this list is not exhaustive and includes proteins that can be found in multiple types of membraneless organelles or biological condensates [69, 70]. While other RNA-binding moieties, including RRM domains, can direct proteins to these protein/ RNA organelles, RGG/RG repeats are critical for recruitment of many of these proteins to membraneless organelles [71, 72]. Specific examples include the RGG region of FMRP directing the protein to cellular cytoplasmic granules [73, 74] and the RGG/RG regions of FUS promoting stress granule localization in cells when the nuclear localization signal is disrupted [65, 75].

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

Purified RGG-Containing Proteins Can Phase Separate Membraneless organelles or biological condensates are thought to form through the process of liquid–liquid phase separation (LLPS) [12, 53, 76–78], by which separate protein-enriched and protein-depleted phases emerge. In many cases, the protein-enriched phase is liquid, although other phases including hydrogels and amyloid fibrils can also be formed by many of the same proteins [79–82]. Formation of separate protein phases can be demonstrated in vitro using purified protein, without addition of RNA, indicating that protein–protein interactions can drive their assembly [53, 83]. The purified RGG/RG region of Laf-1 was shown to be necessary and sufficient for phase separation of the protein [83]. Similarly, the purified RGG-containing regions of FMRP and the disordered portion of Ddx4 can phase separate in vitro [53, 74, 84]. Although not required, RNA has been shown to significantly enhance the ability of a subset of these proteins to undergo RGG region-dependent phase separation [52, 82, 85]. PGL-3 can phase separate in the presence of total C. elegans RNA, but this is disrupted by mutating the RGG repeats [52], providing evidence that RNA binding contributes to the phase separation. The ability of RNA to phase separate in the absence of protein [86] likely contributes to this enhancement. RGG regions and long RNA segments both have multiple recognition elements for each other and mixing them results in a highly multivalent system with protein:protein, protein:RNA and RNA:RNA interactions all possible. Multivalency is a key requirement for phase separation [19], driving the assembly into a protein/RNA-enriched phase. Multivalency is likely an important factor contributing to the enrichment of longer mRNAs in stress granules [87]. Propensity for phase separation and its requirement for RNA likely depend on the amino acid composition and sequence of individual RGG/RG-containing proteins, including the presence of negatively charged residues (see below). Arginines and Glycines: Relevance for Phase Separation

other amino acids. Notably, π–π stacking interactions are compatible with other interaction modes including hydrogen-bonding and electrostatic interactions. As with arginine:RNA interactions, we can obtain snapshots of what these dynamic RGG interactions might look like by scanning the PDB for examples (Fig. 4). Due to the absence of a side chain, glycine residues have an exposed backbone peptide bond and greater range of motion, which promote π–π interactions [21] and the presence of glycine also increases the π–π contact frequency of neighboring side chains, likely due to effects on backbone flexibility [21]. Diglycine peptide bonds can form π-stacking arrangements with other diglycine peptide bonds (Fig. 4a), with arginine guanidino groups (Fig. 4b) and with aromatic side chains (Fig. 4c). In addition to being found in RGG/RG motifs, glycines are also found in phase-separating FG repeats in nucleoporins and in elastin VPGXG repeats [22, 88]. Arginines, despite their repulsive positive charge, can stack on each other via π–π interactions [21] (Fig. 4d) or on aromatic residues in cation–π stacking arrangements (Fig. 4e). Arginines can also form hydrogen bonds via their side-chain guanidino group (Fig. 4f). Experiments in mammalian cells support the importance of the arginines for phase separation. Mutating the arginines in the RGG motifs of Lsm4 to lysines impairs P-body accumulation, probably because lysines are unable to form π–π stacking interactions via their side chains, although arginine dimethylation (see below) also seems to be required in this case [9]. Other arginine-containing polypeptides including SR repeat containing proteins and GR and PR dipeptide-repeat polypeptides (associated with C9orf72 repeat expansions) localize to nuclear membraneless organelles, either nuclear speckles or nucleoli [84, 89]. In vitro experiments indicate that SR repeat proteins and GR and PR repeat polypeptides can also selectively interact with hydrogels formed by hnRNPA2 [84, 89] and PR repeat polypeptides have been demonstrated to form liquid droplets [84]. Thus, arginine and glycine content are strongly correlated with phase separation, with their ease in forming π–π contacts one significant contributor to the ability of RGG/RG motif regions to phase-separate. Charge Interactions in RGG Phase Separation

The specific interactions thought to promote phase separation of RGG are similar to those involved in RNA binding. They include electrostatic interactions (see below), cation–π, π–π and hydrogen-bonding interactions. Arginine and glycine have unique properties that contribute to their relevance for phase separation [21, 84], with both having the potential to form long-range π–π stacking interactions, an interaction mode that can be used to predict phase separation propensity [21]. A survey of the PDB revealed that, in addition to tyrosine and phenylalanine, arginine and glycine residues form π–π interactions as a higher fraction of their total contacts relative to the

As these arginine-containing polypeptides are highly positively charged, it was proposed that counteranions in the buffer, such as phosphate, contribute to stabilizing interactions [84]. The role of buffer counteranions can be substituted by internal negative charges. In the case of Laf-1, the region shown to phase separate (aa1–168) also contains 20 negatively charged residues. The distribution of charges is also important. Ddx4 contains charge blocks, with multiple negative or positively charged residues grouped together. Distributing the charges evenly across the sequence reduces the propensity for phase separation

Please cite this article as: P. A. Chong, et al., RGG/RG Motif Regions in RNA Binding and Phase Separation, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.014

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Review: RGG/RG Motif Regions

Fig. 4. Snapshots of arginine and glycine interactions. Atoms are colored by atom type, with oxygen in red, phosphate in orange, nitrogen in blue and carbons from separate chains in either green or cyan. Rods are used to illustrate planar π–π stacking interactions. Rods are placed normal to the planar interaction, extend to a carbon VDW radius of 1.7 Å and are colored by category with amino acid side chain groups in purple and backbone in darker cyan. Hydrogen bonds are shown with yellow dashed lines. For clarity, not all atoms are shown. (a) Interchain π-stacking arrangement formed by diglycine repeats found within an RGG and an SGG repeat (PDB: 1w74). (b) RGG diglycine backbone to RG guanidino π-stacking/ hydrogen-bonding arrangement (PDB: 1jb0). (c) Diglycine peptide bond forming a π-stacking arrangement with a phenylalanine side chain that is stabilized by strand-pairing and hydrogen bonding (PDB: 2c3b). (d) Arginine residues forming interchain π-stacking arrangement and electrostatic interactions with side chain carboxylate group (PDB: 2yps). (e) Arginine–glycine dipeptide forming aromatic π-stacking/cation–π interactions via the side chain guanidino group and the RG peptide bond (PDB: 1ii2). In this case, the tyrosine residue comes from the same chain as the RG, but is colored cyan for clarity. (f) Arginine guanidino group forming hydrogen-bonding arrangement with backbone oxygens and one serine side chain hydroxyl (PDB: 1qi9).

[53], an observation that can be modeled using analytical theory [90–92], highlighting the role of charge patterning in phase separation. RNA is an important complex counteranion, which, as mentioned above, promotes phase separation of some RGG/RG repeatcontaining proteins. Phosphate groups added via phosphorylation can also serve as counteranions [74] and negatively charged PAR has been demonstrated to promote phase separation of the FET proteins [29]. Interestingly, FET protein RGG repeats have been suggested to be PAR sensors allowing the FET proteins to accumulate at sites of DNA damage, where PAR levels are elevated [29]. Thus, in addition to π–π interactions, electrostatic attraction between the numerous arginines in RGG/RG motif containing regions and negatively charged residues, RNA molecules and other counteranions contributes to drive phase separation. RGG Multivalency and Dynamic Interactions An important feature of RGG/RG motifs is that they often occur in multiple repeats (Table 1), which is relevant for phase separation [19], as discussed previously, with the multivalency of interactions (protein:protein, protein:RNA and RNA:RNA) correlating with phase separation propensity [19, 81, 86]. RG and RGG motifs can occur as many as 35 and 22 times,

respectively, in a single protein chain. Oligomerization regions/domains can increase the number of RGG/RG instances even further. For example, the LSM domain of Lsm14a often forms a six or seven member ring [93]. Since the Lsm14a sequence contains 10 RGG and an additional 7 RG motifs, the Lsm14a complex contains as many as 119 RGG/RG motifs. Multivalency is similarly important for RNA binding. Although it is possible for a short RGG-containing peptide to bind to RNA (i.e., FMRP:sc1), evidence is now accumulating that multiple RGG/RG motifs are generally required for high-affinity binding of RNA. This indicates that the interaction of an individual RGG repeat with RNA is usually weak and suggests a significant role for avidity/multivalency. Deletion of 5 out of 9 RGG repeats in the C-terminus of nucleolin reduced affinity for a presumed four-stranded Gquadruplex DNA structure derived from ETS-1 from 3.3 nM to greater than 40 nM [18]. EWS bound to a Gquadruplex DNA oligonucleotide derived from telomers (Htelo) via its RGG3 region (12 RGG repeats), but not its RGG2 (2 RGG repeats) or RGG1 region (6 RGG repeats) in electrophoretic mobility shift assay studies [17]. In addition, mutating just two of the arginines in RGG3 to lysines inhibited binding of G-quadruplex DNA and mutating four of the arginines in RGG3 eliminated binding [17]. The data support the view that, in general, individual RGG/RG motifs bind relatively

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weakly to RNA, but when linked together can generate a high-affinity interaction. Analysis of which RNAs can segregate into cellular membraneless organelles or are bound by RGG/RG repeat-containing proteins in cells (often in the context of RNP bodies) reinforces the role of multivalency with multiple binding sites. Pull-downs of RNA cross-linked to HA-tagged FET proteins, which contain RRM and Zn-finger domains in addition to their RGG regions, did not reveal a specific RNA recognition element [94]. Transcripts were often bound at multiple sites and there was a significant overlap in the RNA bound by the three different FET proteins [94], despite significant differences in their RGG regions. Work on PGL-3 supports the hypothesis that proteins with multiple RGG repeats are able to bind to most mRNA molecules [52]. The main driver for RNA selection by stress granules (SG), which are enriched in RGG/RG repeat-containing proteins, may in fact be length, which suggests that the number of binding sites may be more important than their specific sequence [52, 87]. Notably, SGlocalized mRNAs have an average length of 7.1 kb versus SG-depleted mRNAs with an average length of 2.5 kb [87]. Intramolecular and intermolecular RGG/RG interactions and intermolecular RGG/RG:RNA interactions in phase-separated states are expected to be highly dynamic, as has been seen for other interactions involving IDPs/IDRs [95]. For example, the interaction between the IDP Sic1 and Cdc4 involves multiple phosphorylated sites on Sic1 interacting with Cdc4 in a dynamic equilibrium, with only local transient ordering around each Sic1 site [96]. Multiple binding sites with weak affinity are characteristic of proteins involved in LLPS [97] but, unlike the Sic1:Cdc4 case in which one partner (Cdc4) has a single dominant binding site, both the RGG/RG proteins and RNA have multiple binding sites. In vitro, multivalency enables interactions with multiple partners, driving phase separation [53, 97]. If the component interactions are weak, then the proteinenriched phase retains liquid properties [97], although viscosity is often 1000-fold or more higher than the viscosity of water (reviewed in Ref. [5]). Higher-affinity, less transient interactions can lead to more viscous gel-like protein-rich phases, fibers or protein aggregates [79, 80, 97]. Fluorescence recovery after photobleaching has been used to confirm that RGGcontaining proteins can move within membraneless organelles and between the protein-rich and proteindepleted phases [52, 75, 98]. Addition of RNA can modulate the viscosity of these droplets [83], presumably by competing with protein–protein interactions. NMR data on several phase-separated RGGcontaining proteins confirm that proteins can remain dynamic in the condensed phase with transient, distributed interactions [74, 99, 100]. In addition to evidence that low-affinity, high-valency interactions are important for retaining liquid properties, there is evidence that ATP-driven enzymatic processes can

be required for maintaining the dynamic behavior in cells [70, 101]. In summary, RNA interactions with these phase-separating RGG/RG repeat-containing proteins are likely driven by low-affinity, high-valency interactions, which also promote phase separation and dynamic behavior of phase-separated cellular bodies.

RGG/RG Methylation Identification of Methylation Sites The discovery of RGG/RG repeats coincided with the discovery that many arginines in RGG/RG motifs are extensively methylated. Asymmetric dimethylarginine (aDMA) was observed in an heterogenous nuclear ribonucleoprotein (hnRNP) homologous protein purified from the slime mold Physarum polycephalum. Amino acid analysis of this slime mold protein indicated that roughly half of all the arginines in the protein were present as aDMA [102]. Purification of nucleolin from hepatoma cells and tryptic digest of the protein yielded a 53-amino-acid peptide. Sequencing of this peptide by Lischwe et al. [3] demonstrated that it contained 10 arginines in RGG repeats, which were all present as aDMA. Remarkably, no monomethylarginine or unmethylated arginine was detected, indicating that these arginines are uniformly asymmetrically dimethylated. Similar results were documented for fibrillarin [3, 4]. More recently, Lsm14A was shown to be asymmetrically dimethylated on one fourth of its arginine residues by amino acid analysis [11] and mass spectrometry provided evidence for aDMA at all observed RGG motifs within the second RGG repeatcontaining region [11]. Methyl groups on arginines of myelin have a half-life of months in rat brains [103], although the location and structure of the myelin may stabilize the methyl groups. In cells, aDMA may be less stable; pretreatment of HeLa cells for 24 h with the methylation inhibitor adenosine-2,3-dialdehyde (AdOx) resulted in a visible reduction in staining using a polyclonal antibody that recognizes aDMA [104]. The pattern of extensive and apparently uniform methylation points to the efficiency of the methylating enzymes and originally suggested that this modification may have only a modest regulatory role. The methods that were used to analyze methylation in these early studies were laborious, since they involved purification of large amounts of protein for each protein under study. While they led to quantitative results for purified proteins, there is the caveat that methylated RGG/RG repeat-containing proteins might be easier to purify than their unmethylated counterparts. More recent papers generally make use of mass spectrometry, which allows for analysis of a greater number of proteins but makes quantification more difficult. These mass spectrometry experiments still require specialized approaches and purification of small subsets of proteins for in-depth analysis and

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10 unambiguous assignment. Methylation sites can be missed in mass spectrometry studies if database searches mis-assign methylated peptides [105], because the mass change upon methylation is found for many other changes, including amino acid substitutions. Dimethylation may also inhibit trypsin digestion, which could prevent digestion and detection of methylated RGG-rich peptides [106]. As is the case with amino acid analysis, mass spectrometry can distinguish between symmetric and asymmetric dimethylation of arginines [107]. These experiments have enabled more extensive analysis of arginine methylation, providing insight into the context of arginine methylation and evidence for differences in the extent of methylation in different cell types, consistent with a more significant regulatory role for methylation. Using these mass spectrometry approaches, Ong et al. [105] were able to demonstrate arginine methylation in over 30 proteins, with the majority being within RG motifs. More than 200 proteins modified by arginine methylation have now been identified [108]. RG motifs represented 71% of identified methylated arginine sites in one study [109]. Mass spectrometry corroborates the high efficiency of arginine methylation at RGG positions. Methylation was observed at 29 out of 30 RGG motifs in EWS [110] and at 20 out of 24 RGG sites in FUS [107]. Although RG motifs represent a huge majority of the methylated peptides, methylation of GR, RA and even PRP containing peptides is also observed [105]. In mammalian cells, arginine methylation can be carried out by at least nine different arginine methylases, including protein arginine methyltransferases (PRMT) 1 through 9 (see Refs. [111, 112] for excellent reviews). RGG/RG motifs are asymmetrically dimethylated by at least three of these enzymes, including PRMT1, 6 and 8 [111, 113]. Other PRMTs are able to modify arginine with symmetric dimethylation [114]. Asymmetric arginine methylation of arginine is also known to occur in insect cells [115], yeast [116] and even in rabbit reticulocyte lysate during in vitro transcription/translation [117] but, notably, is not found on proteins purified from Escherichia coli. Whether methylation functions as a removable regulatory modification in cells is still controversial. Arginine demethylases have been reported in the literature, including KDM3A, KDM4E, KDM5C [118] and JMJD6 [119]. However, these demethylase enzymes have also been identified with other roles, such as lysine demethylation or lysine hydroxylation [118, 120], making it difficult to determine the relative physiological significance of arginine demethylation. In this regard, the ability of JMJD6 to demethylate arginines has been disputed [121, 122]. Interestingly, JMJD5 and JMJD7 remove methylated arginines by proteolytically cleaving off histone tails [123]. Further work on putative demethylase enzymes will be required to resolve these issues. What is clear is that methylation of RGG repeats is not as uniform as suggested by early experiments

Review: RGG/RG Motif Regions

performed on nucleolin and fibrillarin. For example, although five or six arginine residues in the RGG/RG motifs of hnRNPA1 and A3 are asymmetrically dimethylated, respectively, the closely related hnRNPA2 was found to be dimethylated at only one of six arginines in the RGG box region (R254) in rat brain lysate [124]. Furthermore, hnRNPA2 is differentially methylated in different cell types. R254 is primarily dimethylated in rat brain lysates, but there were significant amounts of arginine and monomethylarginine in hnRNPA2 purified from immortalized cell lines [124, 125]. In vitro, hnRNPA2 can be methylated at all four RGG positions by PRMT1 [100]. Mutating 3 out of the 9 arginines in RG motifs in FMRP resulted in a 72% reduction in methylation of FMRP expressed in murine L-M(TK-) cells, suggesting that less than half of the RG motifs are methylated [126]. These differences in methylation levels suggest that some motifs are protected from methylation or that some RGG motifs are preferentially recognized by the methylating enzymes, although the evidence from hnRNPA2 suggests that the latter reason is less likely. Protection against methylation by a high-affinity synthetic RNA ligand has been demonstrated for yeast Hrp1p [127]. Thus, asymmetric dimethylation is less uniform than thought initially and may play a regulatory role. Effect of Methylation on Nucleic Acid Binding Although asymmetric dimethylation of arginine has no effect on the net charge of the residue, it does appear to modestly change the electrostatic properties of the protein. Notably, methylation of only three arginines in hnRNPA1 changes the apparent isoelectric point by a measurable amount on isoelectric focussing gels [128]. This effect could be significant in the context of RGG-repeat binding of negatively charged ligands. Rajpurohit et al. [128] noted that methylation of hnRNPA1 decreased the amount of salt required to elute the protein from a ssDNA-cellulose column. Methylation of arginine has also been suggested to enhance interactions with aromatic groups (study used tryptophan as a test case) by increasing the surface area of the guanidinium moiety available for hydrophobic and stacking interactions and by spreading the positive charge over a greater area [129]. Methylation may also interfere with hydrogen bonding and may introduce steric clashes that inhibit RNA binding. Despite having a structure of the FMRP:sc1 complex, it is difficult to predict the effect on affinity even in this case since little is known about the specific rotameric positions of the guanidino group of arginine in the dimethylated state and how this might affect hydrogen bonding and π-stacking interactions. Methylation of FMRP has alternately been shown to modestly reduce or to not affect its affinity for the sc1 G-quadruplex RNA [74, 126]. Methylated hnRNPA1 was nearly twofold more sensitive to tryptic digestion than unmethylated hnRNPA1 in the presence of a

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ssRNA, implying that the RNA binds more tightly to the unmethylated form of the protein [128]. Methylated and unmethylated yeast Hrp1p bind with nearly the same high affinity to UAUAUA-containing RNAs, although it should be noted that Hrp1p has two RRM domains that were present alongside the RGG repeats in these binding experiments [127]. Methylation of hnRNPK by PRMT1 on six arginines in the RGG repeat region also had no effect on RNA binding; however, hnRNPK also has RNA binding K homology (KH) domains [130]. In contrast, methylation of the RGG3 domain of EWS by PRMT3 resulted in undetectable binding to a Gquadruplex forming DNA in electrophoretic mobility shift assay experiments. However, the methylated protein was still able to bind to a mutant form of the same DNA molecule, which does not fold into the Gquadruplex structure [17]. Thus, the effects of methylation on RNA or DNA binding can range from none to the elimination of binding, depending on the protein and the nucleic acid structural propensity. Arginine Methylation and Nucleocytoplasmic Localization Besides modulating RNA binding, arginine methylation has an effect on nuclear and cytoplasmic localization of RGG/RG repeat-containing proteins. The mechanism may involve changes in the interactions between these proteins and nuclear transport receptors. Unmethylated FUS is able to bind to the nuclear transport receptor, Transportin-1, but the association is inhibited by methylation [131]. Methylation has been proposed to enhance nuclear export of several proteins including Sam68 [132], yeast Npl3p [133], yeast Nab2p [134] and Xenopus CIRP2 [135]. For other proteins, including hnRNPK and CNBP, loss of PRMT methylation activity had no effect on nucleocytoplasmic localization [130, 136]. Methylation appears to affect transport in and out of the nucleus in a manner that varies depending on the experimental system. For example, cytoplasmic levels of hnRNPA2 have been shown to either increase or modestly decrease in response to inhibition of methylation by AdOx [137, 138]. Mutation of the sole dimethylated arginine of hnRNPA2, R254, had no effect on its nucleocytoplasmic localization [124]. Similarly contrasting results were observed for FUS. Treatment with AdOx or depletion of PRMT1 restores nuclear localization to a FUS mutant that is mislocalized to the cytoplasm [131, 139, 140]. However, concurrent shRNA knockdown of PRMT1 and FUS transfection promotes cytoplasmic localization of mutant FUS [140], possibly because there is a significant amount of PRMT1 left in the cells. Overexpression of PRMT1 has also been shown to slightly enhance cytoplasmic localization of wild-type FUS in some cell types [141]. Cytoplasmic levels of the FUS homolog-related TAF15 increase in response to AdOx [142]. Deciphering the role of RGG region methylation in nucleocytoplasmic

localization is complicated by several factors. Many of the proteins involved have nuclear localization signals in addition to their RGG/RG repeat-containing regions [61, 143–146]. Second, use of AdOx or deletion of PRMT1 affects many proteins in the cell. In addition, turnover of methyl groups on arginines is likely quite slow and the level of methylation following inhibition of PRMT1 likely varies. Finally, mutation of arginine residues leads to the loss of potential contacts and may influence localization. Arginine Methylation and Phase Separation Since arginine is significantly correlated with phase separation [21, 84], it was suspected that arginine methylation would affect the phase separation properties of RGG/RG repeat-containing proteins in vitro. Methylation of Ddx4 by PRMT1 yielded protein with an average of 5 or 6 aDMA residues, which lowered the phase transition temperature (reduced the propensity for phase separation) by 25 °C [53]. Likewise, methylation of hnRNPA2 by PRMT1 yielded protein with 4 aDMA residues, disrupted interactions formed by these arginines and raised the threshold concentration required for phase separation [100]. Methylation of the RGG region of FMRP also inhibits droplet formation in vitro [74]. Recent work on FUS also provides evidence that arginine methylation inhibits phase separation [147] or causes phase separated FUS to be more fluid, as opposed to gel-like [148]. Thus, in vitro results indicate that arginine methylation inhibits phase separation or weakens intermolecular interactions. In contrast to these in vitro results, methylation effects in cells are more variable. Methylation is required for Lsm14a and its yeast homolog Scd6 to localize to Pbodies in cells [10, 11]. Depletion of PRMT1 was demonstrated to lead to a marginal decrease in aDMA for Lsm14a that was accompanied by a loss of Lsm14a from P-bodies [11]. Similar results were observed for Lsm4 but in the context of symmetric dimethylation. PRMT5 inhibition led to a decrease in symmetric dimethylation of Lsm4 and reduced visible P-bodies in transformed cells [9]. Likewise, inhibition of dimethylation inhibits incorporation of FXR1P into stress granules, possibly because it reduces binding to FMRP [104]. While methylation seemed to affect FMRP localization to stress granules, it was not directly correlated [104]. Methylation is not required for incorporation of FUS into stress granules either, since both methylated and unmethylated forms can be found in these granules [75]. A factor not considered in this review is the role of Tudor domains, which bind to methylated arginine, including asymmetrically dimethylated arginine [149], and have been shown to attract proteins with methylated arginines to phase-separated bodies, such as nuage [150]. Thus, while methylation of some purified proteins reduces their propensity to

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phase separate in vitro, the role of methylation in directing proteins to membraneless organelles within cells is more complicated.

Conclusion RGG/RG repeats are important mediators of protein: protein and protein:RNA interactions that are critical for formation of membraneless ribonucleoprotein granules. Arginine side chains can form electrostatic, hydrogenbonding, hydrophobic, π–π and cation–π interactions with protein and nucleic acid groups, which drive phase separation and interactions with DNA and RNA. Glycine backbone amide groups also hydrogen bond and form π–π stacking interactions with protein and RNA. Although RGG regions can recognize G-quadruplex structures with high affinity, they likely more often bind within multivalent, dynamic and lower-affinity complexes to long RNA molecules in membraneless organelles that function in RNA processing, such as stress granules. Asymmetric dimethylation of these RGG/RG repeats plays an important though complex role in RNA binding, phase separation and nucleocytoplasmic localization. Since asymmetric dimethylation is so common, these effects will need to be examined in more detail. Another area deserving of more study is the effect of citrullination of arginines on the behaviour of RGG/RG motif containing proteins. Recent data suggest that conversion to citrulline reduces phase separation and aggregation propensity [148, 151]. Greater knowledge of RGG/RG repeats and their post-translational modifications is required for fundamental understanding of sub-cellular organization and its relevance for cancer, neurodegenerative diseases and metabolic diseases [111].

Acknowledgments The authors wish to acknowledge valuable discussions with Brian Tsang, Michael Nosella and Dr. Tae Hun Kim, as well as funding to J.D.F.-K. from the Canadian Institutes of Health Research (114985) and Natural Sciences and Engineering Council of Canada (06718). Received 20 March 2018; Received in revised form 2 June 2018; Accepted 6 June 2018 Available online xxxx Keywords: phase separation; ribonucleoprotein granules; GAR; asymmetric dimethylation; intrinsically disordered

Abbreviations used: aDMA, asymmetric dimethylarginine; AdOx, adenosine2,3-dialdehyde; EWS, Ewing's sarcoma protein; FET, FUS, EWS, TAF15; FMRP, fragile X mental retardation protein; FUS, fused in sarcoma; GAR, glycine–arginine rich; G3BP1, Ras GTPase-activating protein-binding protein 1; hnRNP, heterogenous nuclear ribonucleoprotein; IDP, intrinsically disordered protein; IDR, intrinsically disordered region; LLPS, liquid–liquid phase separation; PAR, poly(ADP-ribose); PDB, Protein Data Bank; PRMT, protein arginine methyltransferase; SERBP1, SERPINE1 mRNA-binding protein 1; TAF15, TATA binding associated factor 15.

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