A functional role for the surface of protein and RNA-protein condensates

Frédéric Allain

Insights into liquid droplet formation via liquid-liquid phase separation and the subsequent liquid-to-solid phase transition are important for understanding cell dynamics, as well as a number of neurodegenerative disorders. We are using the example of the FUsed in Sarcoma (FUS) protein, to investigate the liquid droplet maturation process combining solution- and solid-state NMR spectroscopy, Raman spectroscopy, and light and electron microscopies. Our study (Emmanouilidis et al, Nat. Chem. Biol. 2024) reveals that the surface of the droplets plays a critical role in this process. Indeed, when comparing a biphasic sample, in which liquid droplets are stabilized in an agarose matrix, with a pure monophasic condensed phase sample, we find that the liquid-droplet maturation kinetics is faster in the biphasic FUS sample, owing to the larger surface-to-volume ratio. In addition, using Raman spectroscopy, we observe structural differences upon liquid-droplet maturation between the inside and the surface of liquid droplets, which is of β-sheet content as revealed by solid-state NMR. This is detected very early on and increases over time. In agreement with these observations, a solid crust-like shell is visually seen by microaspiration experiments. After several months, electron microscopy reveals that the matured FUS droplets have converted into solid linear fibrils distinct from the fibril core of seeded fibrils reported previously, as arginine side-chains from the arginine-and-glycine-rich domain (RGG) motif are partially rigidified, highlighting the participation of this motif in the liquid-to-solid transition. In presence of RNA, this aging process is not taking place.

This presentation reveals the critical role of the droplet surface in the maturation of the droplet. More recently, we developed an NMR method to characterize the physical properties of protein-only and protein-RNA condensate and in particular their dynamic properties (diffusion constants and exchange rates). Finally, I will show recent evidence that RNA binding can modify the organization of multiprotein condensates which could have implication for splicing regulation.