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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Biomolecular condensates regulate cellular functions by selectively concentrating molecules.
  • Understanding client partitioning requires studying interactions with both folded and unfolded client states.
  • The FUS RNA Recognition Motif (RRM) is a client protein, and CAPRIN1 is a scaffold protein involved in condensate formation.

Purpose of the Study:

  • To investigate the atomic-level interactions between folded FUS RRM and CAPRIN1 during phase separation.
  • To determine how these interactions mediate the selective enrichment of FUS RRM in CAPRIN1 condensates.
  • To explore the impact of CAPRIN1 post-translational modifications on client partitioning.

Main Methods:

  • Solution Nuclear Magnetic Resonance (NMR) spectroscopy, including [1H-15N]-HSQC, intermolecular Nuclear Overhauser Effect (NOE), and paramagnetic relaxation enhancement (PRE).
  • Chemical shift perturbation analysis to map interaction sites.
  • Investigating FUS RRM partitioning in CAPRIN1 condensates at 40 °C.

Main Results:

  • At 40 °C, ~40% of FUS RRM remains folded within CAPRIN1 condensates, allowing for high-resolution NMR studies.
  • Identified specific interaction surfaces on FUS RRM that bind to CAPRIN1's aromatic- and arginine-rich regions.
  • Observed a 30-fold enrichment of FUS RRM in condensates, with overlapping binding sites for scaffold-scaffold and scaffold-client interactions.
  • Tyrosine phosphorylation of CAPRIN1 significantly disrupted FUS RRM binding and reduced partitioning by over 100-fold.

Conclusions:

  • Folded FUS RRM engages in specific heterotypic interactions with CAPRIN1, driving its selective partitioning into biomolecular condensates.
  • Scaffold-client and scaffold-scaffold recognition share common interaction interfaces, suggesting a unified mechanism for condensate assembly.
  • Post-translational modifications, like tyrosine phosphorylation of CAPRIN1, act as critical regulators of condensate composition by modulating client interactions.