Controlled liquid-liquid phase separation via the simulation-guided, targeted engineering of the RNA-binding protein...

Area of Science:

• Plant molecular biology
• Biophysics
• Biochemistry

Background:

• Phloem-Associated RNA-Chaperone-Like (PARCL) is a plant-specific RNA-binding protein (RBP) abundant in phloem.
• PARCL forms large, mobile biomolecular condensates in the phloem, suggesting a role in RNA transport.

Purpose of the Study:

• To identify the molecular drivers of PARCL phase separation.
• To validate the role of specific residues in PARCL condensation and RNA binding.

Main Methods:

• Coarse-grained molecular dynamics simulations to generate a residue interaction map.
• Site-directed mutagenesis of candidate residues.
• In vitro and in vivo experiments for validation.
• Simulations incorporating microRNA to study RNA-protein interactions.

Main Results:

• A residue interaction map identified candidate residues critical for PARCL phase separation.
• Mutagenesis simulations showed disrupted condensation, confirming the role of identified residues.
• Experiments validated the simulation predictions regarding PARCL condensation.
• A specific PARCL region was identified to interact with microRNA, consistent with bioinformatics and experimental data.

Conclusions:

• Specific amino acid residues are crucial for driving PARCL phase separation and condensate formation.
• The findings provide a foundation for model-guided engineering of PARCL-based biomolecular condensates.
• Understanding PARCL's phase separation mechanism offers insights into RNA transport in plant phloem.