Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Abstract

Macromolecular phase separation is thought to be one of the processes that drive the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation, especially at low endogenous concentrations found in cells, are thought to follow the tenets of classical nucleation theory describing a sharp transition between a dense phase and a dilute phase by dispersed monomers. Here, we used in vitro biophysical studies to study subsaturated solutions of phase separating RNA binding proteins with intrinsically disordered prion like domains (PLDs) and RNA binding domains (RBDs). Surprisingly, we find that subsaturated solutions are characterized by heterogeneous distributions of clusters comprising tens to hundreds of molecules. These clusters also include low abundance mesoscale species that are several hundreds of nanometers in diameter. Our results show that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. Interestingly, however, cluster formation and phase separation can be decoupled from one another using solutes that impact the solubilities of phase separating proteins. They can also be decoupled by specific types of mutations. Overall, our findings implicate the presence of distinct, sequence-specific energy scales that contribute to the overall phase behaviors of RNA binding proteins. We discuss our findings in the context of theories of associative polymers.

Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates

“…They may thus play an important role for functional and pathological LLPS of Tau. Recently, similar mesoscopic clusters were reported for p53 in cells (Yang et al, 2021) and for RNA binding proteins in vitro (preprint: Kar et al, 2022).…”

Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales

“…Moreover, future studies must include the spherical Bre1 shell as a boundary condition which presumably constrains the 3D-orientation of the Lge1 C-terminus and thereby impinges on the geometry of the Lge1 meshwork. Finally, the fractal model predicts a coexistence of differently sized clusters within a condensate, as reported recently 61 , which have a characteristic scaling of mass with condensate size in the nm to μm range. It will be important to analyze to what extent these features are retained in more complex, biologically relevant contexts.…”

Controlling cluster size in 2D phase-separating binary mixtures with specific interactions

“…We show that such size-control process, although it can lead to stationary states within observation time (Fig. 6), is not of obvious thermodynamic origin, as instead recently found for analogous processes in different systems 19,52 . On the contrary, it can be driven by a kinetic trap whose depth increases with bond strength and unevenness in concentrations.…”