In Vitro Phase Separation Characterization of the Arabidopsis thaliana Glycine-Rich RNA-Binding Protein AtGRP2.

The Arabidopsis thaliana glycine-rich RNA-binding protein 2 (AtGRP2) belongs to the cold-shock domain (CSD) protein family and regulates flowering and abiotic stress responses. It comprises an N-terminal RNA recognition motif (RRM) and a C-terminal intrinsically disordered glycine-rich domain (GRD) implicated in phase separation (PS). Herein, we investigated the PS behavior of AtGRP2 in vitro and its modulation by RNA. PS of recombinant AtGRP2 was monitored by fluorescence spectroscopy and turbidity measurements. Under crowding conditions, the full-length protein formed spherical, micron-sized droplets with low fusion dynamics, indicating a viscoelastic state. These condensates were resistant to high salt but disrupted by 1,6-hexanediol, highlighting the role of hydrophobic interactions in PS. AtGRP2 condensation was disrupted in the presence of total Arabidopsis RNA or the Lin28-specific miRNA, a prelet-7g precursor, in a dose-dependent manner. Lin28 is a human counterpart to AtGRP2, sharing similar domains except for the GRD. Prelet-7g was found to partition into the condensates and directly interact with AtGRP2. Mutational analysis showed that the CSD alone was insufficient for PS, while the isolated C-terminal region formed solid aggregates. Deletion of the GRD had little impact on phase behavior; however, removal of the CCHC-type zinc finger or arginine residues completely abolished droplet formation, highlighting their critical role in mediating the multivalent interactions required for PS. These results demonstrate AtGRP2 undergoes PS driven by its disordered C-terminal region and modulated by RNA binding. This mechanism may contribute to its localization to membraneless organelles, including the nucleolus, supporting stress adaptation similarly to Lin28.