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

## TL;DR

This study uses simulations to understand how the plant protein PARCL forms condensates and how it interacts with RNA, offering new insights into biomolecular phase separation.

## Contribution

The study introduces a simulation-guided approach to engineer phase separation in the RNA-binding protein PARCL.

## Key findings

- Key residues involved in PARCL's phase separation were identified through simulations and validated experimentally.
- Mutations in PARCL affect phase separation and RNA recruitment independently.
- MicroRNA influences PARCL's phase separation behavior as shown by simulations and experiments.

## Abstract

The Phloem-Associated RNA-Chaperone-Like (PARCL) protein is a plant-specific RNA-binding protein (RBP) that is highly abundant in the phloem. PARCL has been observed to form large biomolecular condensates that move within the phloem stream, potentially being involved in RNA transport. Here, we present results on unraveling drivers for PARCL’s phase separation. We used coarse-grained molecular dynamics simulations to compute a residue interaction map that identifies candidate residues involved in phase separation. Subsequent simulations with mutations of candidate residues resulted in disrupted condensation, supporting their involvement in phase separation. We performed in vitro and in vivo experiments to validate these predictions. To investigate the RNA-binding of PARCL, we added microRNA to the simulations and identified a short region of PARCL that consistently made contact with the miRNA in agreement with bioinformatics predictions and experiments. We discuss the implications of our findings in terms of model-guided engineering of biomolecular condensates.

•Simulations of Phloem-Associated RNA-Chaperone-Like protein show phase separation•Contact analysis identifies key residues that drive phase separation•Simulations capture the influence of microRNA on PARCL phase separation•Mutations separately affect phase separation and condensates’ miRNA recruitment

Simulations of Phloem-Associated RNA-Chaperone-Like protein show phase separation

Contact analysis identifies key residues that drive phase separation

Simulations capture the influence of microRNA on PARCL phase separation

Mutations separately affect phase separation and condensates’ miRNA recruitment

Molecular dynamics; Biomolecules; Protein; Bioengineering; Biophysics; Computational bioinformatics