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FC0097
Fused in Sarcoma (FUS)  -  FUS


Biological function
FUS is an RNA-binding protein, which localizes both to cytoplasmic RNP granules and transcriptionally active nuclear puncta and is associated with processes spanning transcriptional regulation, pre-mRNA splicing, and mRNA transport and stability. FUS is associated with protein aggregation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as well as chromosomal translocation in certain sarcomas and leukemias.

Domain organization/sequence features
The N-terminal low-complexity (LC) domain of FUS (1–163 AA) is a highly conserved prion-like domain composed primarily of serine, tyrosine, glycine, and glutamine rich and contains only two charged residues. The 24 tyrosine residues are arranged in [S/G]Y [S/G] repeats that are often followed by one to three glutamine or proline residues. The LC domain mediates protein interactions in both nuclear assemblies and cytoplasmic RNP granules. FUS LC drives the aggregation of FUS into protein inclusions in vitro and in models of ALS and FTD.

Structural evidence
13C NMR chemical shifts lack of significant deviation between the observed shifts and those of a random coil reference demonstrate that the monomeric FUS LC domain is highly disordered, lacking significant population of α helices or β sheets. NMR relaxation experiments (15N R2, 15N R1, heteronuclear NOE) also show that the FUS LC domain is uniformly disordered in monomeric form. The lack of large variations in these dynamic observables due to slower rotational diffusion in structured areas indicates that no stable structured subregions of FUS LC are formed. This is also consistent with CD data. In phase separated state, FUS LC yielded observable but broad resonances in the 1H 15N HSQC, suggesting that some local motions are retained in the higher-order assembly. 1H 15N HSQC spectra is highly similar to those of the monomeric FUS LC (50 mM), demonstrating that the global disordered structure is also retained in the phase-separated state. The lack of large chemical shift differences in 1H 13C HSQC spectra between the phase-separated and dispersed phases indicates that no significant conformational changes has occurred and the local structure of the protein also remains disordered in the phase- separated state. Local motions of the peptide as measured by 15N spin relaxation experiments show evidence for restricted mobility compared to that of the monomeric, dispersed protein. Conformational exchange processes, if any, likely occur faster than the millisecond timescale. Upon interaction with RNAP II CTD, two-dimensional NMR spectra show that the CTD signal is nearly uniformly attenuated to ~75% of the control sample, indicating that monomers of the CTD are incorporated into, or interact with, the FUS LC phase- separated state.

Biochemical evidence
FUS LC form granules in vitro, which flow, fuse and spontaneously return to a spherical shape as observed for cytoplasmic RNP granules. Granule formation for FUS LC was weakly dependent on increasing salt concentration (until 150 mM), suggesting that primarily not the electrostatic interactions stabilized the FUS granule. RNA polymerase II CTD can directly interact with FUS LC domain phase-separated states and can nucleate their assembly.

Structure/Mechanism
FUS LC may populate a conformation or state with lifetime line broadening or intermediate chemical shift timescale conformational exchange either within the phase-separated state or at the phase interface. Thus a model emerges when CTD binds FUS LC in its phase-separated state and not as dispersed monomers, but does not establish permanent contacts.

Mechanism category
tethering

Significance
Fuzziness of FUS LC contributes to the elevated dynamics of the RNP granules and enables a rapid exchange with the environment of the droplet. Variable conformations also mediate interactions with RNAP II CTD, without a significant stabilization of any structural states.

Medical relevance
Five missense or short deletion mutations located within the regions coding for FUS LC are linked to ALS, for example G156E, which increases FUS aggregation propensity in vitro and in cell culture. More than a dozen related sarcomas and leukemias are caused by chromosomal translocations fusing the LC domain of FUS or that of two other human paralogs, RNA-binding protein EWS and TATA-binding protein-associated factor 2N (product of the TAF15 gene), to one of several DNA-binding domains, forming strong transcriptional activators.