RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of...

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• The SARS-CoV-2 nucleocapsid (N) protein plays a crucial role in viral RNA packaging and condensate formation.
• Understanding the specific mechanisms of N-RNA interactions is vital for comprehending viral function.
• Distinguishing sequence-independent from sequence-specific RNA binding by the N protein remains unclear.

Purpose of the Study:

• To identify specific RNA structures and N protein domains involved in sequence-specific interactions.
• To elucidate the roles of different N-RNA interactions in phase separation.
• To differentiate between interactions driving viral function and those promoting condensate formation.

Main Methods:

• Utilized fluorescence anisotropy to measure binding affinities of N protein domains (NTD, CTD) to various RNA models.
• Employed nuclear magnetic resonance (NMR) spectroscopy to identify RNA-binding sites on the N protein NTD.
• Conducted phase separation assays using N protein domains and different RNA structures (ssRNA, dsRNA).

Main Results:

• The N-terminal domain (NTD) preferentially binds single-stranded RNA (ssRNA) and is the primary binding site but not essential for phase separation.
• NMR identified a secondary, weaker RNA-binding face on the NTD, prominent with dsRNA or impaired binding.
• Phase separation is promoted by multiple weak interactions (e.g., CTD, NTD secondary face) rather than strong, specific ones.

Conclusions:

• Both strong, specific N-RNA interactions and multivalent weak interactions contribute to the N protein's diverse functions.
• Weak interactions, particularly involving the CTD and NTD's secondary face, are key drivers of liquid-liquid condensate formation.
• The NTD's primary binding site is important for RNA binding, but not solely responsible for phase separation.