Protein-RNA condensation kinetics via filamentous nanoclusters

Author(s)

Peralta-Martinez, Ramon ; Visentin, Araceli ; Salgueiro, Mariano ; Borkosky, Silvia Susana ; Aleixo, Mariana Araujo Ajalla ; Portugal, Rodrigo Villares ; Sanchez, Ignacio Enrique ; Prat-Gay, Gonzalo

• Document type: Journal article
• Source: Protein Science; v. 34, n. 6, p. 14-pg., 2025-06-01.

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. (AU)

• FAPESP's process: 20/06062-1 - Biological complexes in action, in vitro and in silico (Exploiting Big-Data in Cryo-EM)
• Grantee: Marin Gerard van Heel
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 22/05088-2 - Amplifying RI impact with a global perspective: a Regional Hubs model
• Grantee: Rodrigo Villares Portugal
• Support Opportunities: Regular Research Grants