Protein– RNA condensation kinetics via filamentous nanoclusters

Abstract Protein–RNA phase separation is at the center of membraneless biomolecular condensates governing cell physiology and pathology. Using an archetypical viral protein–RNA condensation model, we determined the sequence of events that starts with sub‐second formation of a protomer with two RNAs per protein dimer. Association of additional RNA molecules to weaker secondary binding sites in this protomer kickstarts crystallization‐like assembly of a molecular condensate. Primary nucleation is faster than the sum of secondary nucleation and growth, which is a multistep process. Protein–RNA nuclei grow over hundreds of seconds into filaments and subsequently into nanoclusters with approximately 600 nm diameter. Cryoelectron microscopy reveals an internal structure formed by incoming layers of protein–RNA filaments made of ribonucleoprotein oligomers, reminiscent of genome packing of a nucleocapsid. These nanoclusters progress to liquid condensate droplets that undergo further partial coalescence to yield typical hydrogel‐like protein–RNA coacervates that may represent the scaffold of large viral factory condensates in infected cells. Our integrated experimental kinetic investigation exposes rate‐limiting steps and structures along a key biological multistep pathway present across life kingdoms.