RNA keeps AtGRP2 from phase separating into highly viscoelastic condensates

AtGRP2 is an Arabidopsis thaliana glycine-rich RNA-binding protein that plays crucial roles in plant development and response to abiotic stress. AtGRP2 consists of an N-terminal cold shock domain (CSD) followed by a disordered C-terminal region containing two CCHC-type zinc fingers interspersed with glycine-rich segments. Here, we investigated the liquid-liquid phase separation (LLPS) of AtGRP2 in vitro and its modulation by RNA interaction. An mEGFP-fused construct of full-length AtGRP2 was expressed in Escherichia coli BL21 DE3 and purified by nickel-affinity and size exclusion chromatography. We utilized fluorescence microscopy and turbidity (absorbance at 600 nm) assays to monitor LLPS behavior. We observed that AtGRP2 forms spherical condensates under molecular crowding conditions, regardless of the crowding agent. These condensates exhibited low dynamics and did not undergo fusion, indicating their highly viscoelastic nature. In addition, AtGRP2 condensates were resistant to high concentrations of NaCl, but sensitive to 1,6-hexanediol, suggesting that hydrophobic interactions drive LLPS. We demonstrated that basic pH disassembled the condensates, while acidic pH promoted the formation of network-like aggregates. The addition of total Arabidopsis RNA disrupted AtGRP2 LLPS in a dose-dependent manner. Additionally, the miRNA precursor prelet7g, which is a specific RNA ligand of the human protein Lin28, partitioned into and dissolved AtGRP2 condensates, even in the excess of protein, suggesting the role of RNA binding in maintaining AtGRP2 in a diffuse state. These results indicate that AtGRP2 undergoes LLPS, which may be relevant for its function in membraneless organelles such as the nucleolus, where it may maintain nucleolar integrity in response to abiotic stress, similar to the human protein Lin28.