Abstract
SummaryReversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular β-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.
Highlights
We investigated phase separation in purified full-length FUS proteins in which (1) multiple arginines in the structured C-terminal domain (sCTD) were mutated to alanine or lysine or (2) multiple tyrosines in the LC domain were mutated to alanine or phenylalanine
Given that transportin 1 (TNPO1) is expressed in axon terminals, and may function as a molecular chaperone, we investigated whether modest overexpression of TNPO1 might restore FUS RNP granule function in axon terminals treated with AdOx or expressing ‘‘cation-p enhanced’’ FUS constructs
The experiments described here reveal that phase separation is driven by multivalent cation-p interactions, which occur physiologically between multiple arginines in the sCTD and multiple tyrosines in the LC domain
Summary
Expression and purification of FUS TNPO1, EWS and TAF15 Constructs encoding FUS residues 1-526 and its mutants, LC-mEmerald (aa1-214) and CTF-mCherry (aa215-526), were cloned into pACEBac vector with a TEV cleavable N-terminal MBP tag and an EmGFP or mCherry-6xHis- C-terminal tag. Cell pellets were mixed with the resuspension buffer containing 50 mM Tris, 1 M KCl, 0.1% CHAPS, 1 mM DTT, 5% glycerol at pH 7.4, and proteins purified using three steps purification scheme including, Ni-NTA affinity column, Amylose affinity column followed by size exclusion chromatography in the buffer containing 50 mM Tris, 1 M KCl, 1 mM DTT, 5% glycerol at pH 7.4. Constructs encoding full length human EWS or human TAF15 were cloned into pBACEBac vector with a TEV protease cleavable N-terminal MBP tag. Fusion proteins were subjected to TEV protease cleavage and the MBP tag was further removed by size exclusion chromatography
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