Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa
Basic protein information
Accession P35637
Common name FUS
Gene FUS
Organism Homo sapiens
Uniprot name RNA-binding protein FUS
Basic LLPS information
Organelle cytoplasmic stress granule; cytoplasmic ribonucleoprotein granule
Type of experimental evidence
Protein region(s) mediating LLPS
Full protein sequence contributes to LLPS: PLD and RNA-binding domains (RRMs and RGGs)
Based on the experimental results of the following publication: 29961577
Molecular features viewer
PDB structures
Extended LLPS information
Functional description
FUS is DNA/RNA-binding protein that plays a role in various cellular processes such as transcription regulation, RNA splicing, RNA transport, DNA repair and damage response. There are about 30 FUS family proteins in the human genome. These include FET proteins (FUS and the related proteins EWSR1 and TAF15), TDP-43, and hnRNPA1. Aberrant phase transitions of many FUS family proteins have been associated with the onset of age-related neurodegenerative diseases. The amino acid sequences of FUS family proteins can be divided into two modules: a low sequence complexity domain also known as a prion-like domain (PLD) and a domain that binds RNA (RNA-binding domain, or RBD). The PLD contains a small subset of amino acids including polar residues, such as glycine, glutamine, and serine, as well as aromatic residues, generally tyrosine, while the RBDs comprise one or more folded RNA recognition modules (RNA recognition motif, RRM), but they also carry regions of significant intrinsic disorder. The intrinsically disordered regions in RBDs are typically enriched in glycine and positively charged residues, such as arginine (PMID:29961577). In vitro, some PLDs will drive condensate formation as autonomous units, as a result, there has been a focus on PLDs as the main determinants of phase transitions in cells (PMID:22579282, PMID:26455390, PMID:26412307, PMID:28942918). However it seems that the PLD itself cannot be regarded as a physiologically relevant module of the protein from the point of view of LLPS, since it has been shown later that the phase separation of full-length FUS is mainly driven by cation-pi interactions between the Arg residues of the RBD and the Tyr residues of the PLD. The PLD itself has only few Arg residues and thus its hydrogel formation is rather dependent on pi-pi interactions between the Tyr residues and prion-like behavior, which are largely supressed in the context of full-length FUS. Phase separated condensates of FUS and TAF15 PLDs have been found to be able to recruite the C-terminal repetitive tail of RNA polymerase II, which is especially important in light of the existance of oncogenic fusion proteins harboring those PLDs in combination with DNA-binding domains (PMID:26455390, PMID:24267890). Both phosphorylation and phosphomimetic variants reduce the aggregation-prone/prion-like character of FUS PLD, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Also, phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity (PMID:28790177). FUS can form pathological protein aggregates, and specific mutations in FUS have been identified in patients suffering from neurodegenerative diseases. Mutations in FUS are associated with amyotrophic lateral sclerosis (ALS) and rare forms of frontotemporal lobar degeneration (FTLD). Patient-derived mutations in or around the prion-like domain of FUS (G156E and R244C) have an increased tendency to form aggregates (PMID:26317470). FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. Its recruitment at DNA damage sites happens through its binding to PAR (attached to the DNA as a result of PARP-1 activation), and leads to the assembly of damaged DNA-rich compartments that recruit DNA repair factors (PMID:31067465). ATP and nucleic acids share a common two-stage effect on LLPS of several RBPs, including FUS: enhancement of LLPS at low concentrations but dissolution at high concentrations. Cation-π interactions between the PLD and RBD act as the major driving force for LLPS of FUS. Both ATP and oligonucleic acids modulate LLPS of FUS and its dissected domains in the same manner, primarily by targeting the cation-π interactions through specific binding to Arg/Lys residues, as revealed by NMR (PMID:31188823).
Literature supporting the LLPS: 22579282, 22579281, 26412307, 28942918, 26455390, 28790177, 26317470, 29547565, 26526393, 29677513, 29677514, 29677515, 28041848, 30205960, 29897835, 29961577, 26286827, 31067465, 31188823
Functional class of membraneless organelle: activation/nucleation/signal amplification/bioreactor; protective storage/reservoir
Binding partners (at biological protein concentrations)
1) poly(ADP-ribose) (not required, drives LLPS nucleation in cells) 2) RNA (induces/inhibits droplet formation in low/high concentrations) 3) karyopherin-β2 (weakens LLPS) 4) ATP (induces/inhibits droplet formation in low/high concentrations)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required.
Molecular interaction types contributing to LLPS
cation-π (cation-pi) interactions (PMID:29961577) π-π (pi-pi) interactions (PMID:29961577) electrostatic (cation-anion) interaction (PMID:29961577)
Determinants of phase separation and droplet properties
1) protein concentration of FUS 2) salt concentration 3) crowding agent concentration 4) number of Tyr and Arg residues 5) modification state 6) RNA concentration
Membrane cluster No
Partner-dependent No
RNA-dependent No
PTM required No
Domain-motif interactions No
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
FUS, EWSR1, and TAF15, which constitute the FET family showed robust phase separation (particle size and count by microscopy) in the absence of crowding agents at a protein concentration of 5 μM. Although the majority of the FUS family of proteins can phase separate, only the FET proteins possess the requisite sequence features to drive phase separation at low protein concentrations and physiologically relevant salt concentrations. The measured saturation concentration of full-length FUS is 2 μM in 75 mM KCl. Surprisingly, the FUS PLD (truncated), expressed and purified from insect cells, does not drive phase separation as an independent module even for protein concentrations that are as high as 120 μM. Therefore, the saturation concentration for the PLD is almost two orders of magnitude higher than the saturation concentration for the full-length protein. The RBD alone did not phase separate. However, when the FUS PLD was mixed with the FUS RBD at a ratio of 1:1, the PLD phase separated at concentrations as low as 15 μM. Mutational studies, where all the Tyr residues of the PLD (Y-->S) and/or all the Arg residues of the RBD (R-->G) have been changed suggested that these residues are the major determinants of LLPS of FUS and that their number governs the saturation concentration of FUS both in vitro and in vivo in cells (PMID:29961577). Further mutagenesis studies implied that the driving forces for phase separation in vitro and in vivo, quantified in terms of measured saturation concentrations, follow the order: tyrosine-arginine > tyrosine-lysine > phenylalanine-arginine > phenylalanine-lysine, which shows that the selective preference for tyrosine-arginine interactions cannot be solely due to generic cation-p interactions. Instead, the specific chemical structures of the tyrosine and the arginine side chains appear to be important determinants of the complementarity of tyrosine-arginine interactions (PMID:29961577). In vivo expression of the protein in human cells, truncated to residues 1-211 (low complexity N-terminal region) led to assembly into structures with remarkably spherical morphology via liquid demixing, evidenced by GFP-tagging and fluorescence and phase-contrast microscopy. The RGG repeat region was shown to mediate poly(ADP-ribose) (PAR) binding, using in vivo expression of GFP-tagged full length protein with induced mutations in the RGG region. The number of RGG repeats determined the efficiency of PAR binding, co-localization with PAR, and hence, recruitment to sites of DNA damage. While this is not strictly required for LLPS, PAR binding serves as a nucleation event, increasing local protein concentration at sites of DNA damage. Protein localization and co-localization was followed using standard widefield, confocal, high-content microscopy and time-lapse imaging. In a cellular, in vivo context, depletion of FUS with RNA interference, and abrogation of PAR formation both led to the lack of granule formation at sites of DNA damage, observed using time-lapse bright-field and phase-contrast live-cell microscopy. In vitro cell-free measurements recombinant FUS was incubated with and without sub-stoichiometric amounts of PAR, forming spontaneous aggregates, which were consistently larger in the presence of PAR, followed by transmission electron microscopy (TEM). Consistent with in vivo results, these data provide evidence for the intrinsic ability of PAR chains to nucleate aggregation of low complexity domain-containing disordered proteins (PMID:26286827). In vitro phosphorylation of recombinant, isolated FUS LC reduces its ability to bind hydrogels formed from amyloid‐like fibrils of purified recombinant, unphosphorylated FUS LC (PMID:22579282) Both phosphorylation and phosphomimetic variants reduce the aggregation-prone/prion-like character of FUS PLD, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Also, phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity (PMID:28790177). BAC transgene has been generated for the in vivo expression of GFP-tagged FUS after introduced into HeLa and embryonic stem (ES) cells to generate stable cell lines. Mass spectrometry was used to determine the physiological concentration of FUS in HeLa cells, that is around 2 μM in the cytoplasm and between 4 and 8 μM in the nucleus. Actinomycin D (a potent inhibitor of RNA polymerase II) treatment of FUS-GFP HeLa cells suggested that under normal conditions, FUS assembles into compartments (particle size and count) that may be associated with transcription or splicing. FUS accumulated in the cytoplasm in heat-stressed cells and coalesced into stress granules (in vivo protein localization). In vivo FUS assemblies have all the hallmarks of a liquid state: they turn over quickly; are spherical; and when they fuse, they relax into one spherical assembly. A 10% solution of either dextran or polyethylene glycol (PEG) induced the in vitro assembly of recombinant GFP-tagged FUS (10 μM) purified from insect cells (but not that of truncated FUSΔPLD) into round micrometer-sized structures (PMID:26317470). Inhibition of PARP1/2 prevented the recruitment of FUS to DNA lesions. After 8 hr, no fusion events could be detected for G156E FUS, while 81% of the wild-type FUS droplets still fused. By 12 hr, wild-type droplets also stopped fusing. This indicates that there is indeed a change in the biophysical properties of FUS droplets with time and that the properties of the G156E FUS droplets are changing more quickly than those of the wild-type FUS droplets. This is consistent with the observation that G156E has a higher propensity to aggregate. Another patient-derived mutant of FUS R244C also accelerated the conversion of FUS droplets to a fibrous state (PMID:26317470).
Experimental observations supporting the liquid material state of the condensate
dynamic exchange of molecules with surrounding solvent (PMID:26317470) dynamic movement/reorganization of molecules within the droplet (PMID:26317470) morphological traits (PMID:26317470)