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

Research output: Contribution to journalResearch articleContributedpeer-review


  • Mrityunjoy Kar - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Furqan Dar - , Washington University St. Louis (Author)
  • Timothy J. Welsh - , University of Cambridge (Author)
  • Laura T. Vogel - , Heinrich Heine University Düsseldorf (Author)
  • Ralf Kühnemuth - , Heinrich Heine University Düsseldorf (Author)
  • Anupa Majumdar - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Georg Krainer - , University of Cambridge (Author)
  • Titus M. Franzmann - , Chair of Cellular Biochemistry (Author)
  • Simon Alberti - , Chair of Cellular Biochemistry (Author)
  • Claus A.M. Seidel - , Heinrich Heine University Düsseldorf (Author)
  • Tuomas P.J. Knowles - , University of Cambridge (Author)
  • Anthony A. Hyman - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Rohit V. Pappu - , Washington University St. Louis (Author)


Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.


Original languageEnglish
Article numbere2202222119
JournalProceedings of the National Academy of Sciences of the United States of America : PNAS
Issue number28
Publication statusPublished - 12 Jul 2022

External IDs

PubMed 35787038
ORCID /0000-0003-4017-6505/work/142253850


Research priority areas of TU Dresden

DFG Classification of Subject Areas according to Review Boards

ASJC Scopus subject areas


  • associative polymers, mesoscale clusters, phase separation, sol-gel transitions, stickers and spacers, Biomolecular Condensates, RNA-Binding Proteins/genetics, Biophysics, Mutation, RNA-Binding Motifs

Library keywords