Fused in sarcoma undergoes cold denaturation: Implications for phase separation

Abstract

The mediation of liquid-liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn 2+ , and Ca 2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0 C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.

The prion-like domain of the chloroplast RNA binding protein CP29A is required for cold-induced phase separation next to nucleoids and supports RNA splicing and translation during cold acclimation

“…Several other RNA-binding proteins that exhibit temperature-dependent phase separation, such as Fused in Sarcoma (FUS), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), or the poly(A)-binding Protein1 (Pab1), utilize their RNA-binding domains next to their IDRs to drive phase separation (Molliex et al, 2015; Burke et al, 2015; Yoshizawa et al, 2018; Riback et al, 2017). For example, in the case of FUS, phase separation is supported by the cold denaturation of its zinc finger domain (Félix et al, 2023). Our in vitro and in vivo deletion analyses suggest that for CP29A, the PLD is the dominant factor in mediating cold-induced phase separation.…”

Hydrophobic Homopolymer’s Coil–Globule Transition and Adsorption onto a Hydrophobic Surface under Different Conditions

“…121 Furthermore, recent experiments show that the folded domains of fused in sarcoma (FUS), a protein with low-complexity IDRs, undergo cold denaturation, with implications for its mediation of LLPS. 122 Here, we observe for the hydrophobic polymer under biased confinement the analogous of the cold denaturation at low T and a high enough P (Figure 2), e.g., at (k B T/4ϵ, Pv 0 /4ϵ) = (0.050, 0.3) (Figure 4). This low-T unfolding is energy-driven due to the contribution of the hydration HBs to the TOT and is unaccessible if the energy gain of the HB at the hydrophobic interface is too small (Figure S1).…”

“…The BF model shows that in bulk, at low enough T and appropriate P , the coil–globule transition is reentrant (Figure ) as seen in experiments, at relatively high pressures, in structured proteins, − and unstructured polymers . Furthermore, recent experiments show that the folded domains of fused in sarcoma (FUS), a protein with low-complexity IDRs, undergo cold denaturation, with implications for its mediation of LLPS …”

Intrinsically disordered protein’s coil-to-globule transition and adsorption onto a hydrophobic surface under different conditions

“…99 Furthermore, recent experiments show that the folded domains of fused in sarcoma (FUS), a protein with low-complexity IDRs, undergo cold denaturation, with implications for its mediation of LLPS. 100 Here, we observe for the IDP under biased confinement the analogous of the cold denaturation at low T and a high enough P (Fig. 2), e.g., at (k B T /4ϵ, P v 0 /4ϵ) = (0.050, 0.3) (Fig.…”

“…99 Furthermore, recent experiments show that the folded domains of fused in sarcoma (FUS), a protein with low-complexity IDRs, undergo cold denaturation, with implications for its mediation of LLPS. 100…”