Mass spectrometry of RNA-binding proteins during liquid-liquid phase separation reveals distinct assembly mechanisms and droplet architectures

Phase separation of heterogeneous ribonucleoproteins (hRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility-mass spectrometry, and molecular dynamics simulations. We used a phase separation-compatible spider silk domain and pH changes to control the self-assembly of the hRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with phase separation. We find that NT*-FUS monomers undergo an unfolded-to-globular transition, whereas NT*-TDP-43 oligomerizes into partially disordered dimers and trimers. NT*-hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over phase separation. The divergent assembly mechanisms result in structurally distinct complexes, indicating differences in RNA processing and translation depending on biological context.