AtGRP7 PHASE SEPARATION IS DRIVEN BY ITS C-TERMINAL GLYCINE-RICH DISORDERED REGION

AtGRP7 (Arabidopsis thaliana glycine-rich protein 7) is an extensively researched member of the plant glycine-rich protein family, known for its role in regulating crucial physiological processes such as circadian rhythm, flowering time, and response to abiotic stress. AtGRP7 is an RNA-binding protein composed of an N-terminal RNA recognition motif (RRM) and a C-terminal disordered region enriched in glycines. Due to its specific amino acid sequence bias and disordered structure, we proposed the possibility of AtGPR7 undergoing liquid-liquid phase separation (LLPS), a physical phenomenon underlying the formation of membraneless organelles. To investigate LLPS, we expressed different AtGRP7 constructs (full-length, N-terminal, and C-terminal domains) fused with mEGFP in Escherichia coli BL21 DE3, and then purified them using nickel-affinity and size exclusion chromatography. We utilized fluorescence microscopy and turbidity (absorbance at 600 nm) assays to explore LLPS behavior. Under molecular crowding conditions, mEGFP-AtGRP71-176 formed spherical and micrometric condensates, albeit with slow kinetics (~1h30). These condensates were found to be highly sensitive to NaCl but partially resistant to the aliphatic alcohol 1,6 hexanediol, suggesting that electrostatic interactions play a significant role in LLPS. Basic pH (pH > 7.5) disrupted AtGRP7 condensates, while acidic pH (pH < 5.0) induced a transition to a solid aggregate, as indicated by fluorescence recovery after photobleaching (FRAP) results. The addition of a specific RNA ligand (30 nts) caused the system to become monophasic, suggesting that RNA keeps AtGRP7 in the diffuse state. We further demonstrated that the isolated N-terminal domain (mEGFP-AtGRP71-90) did not undergo LLPS, even at high protein concentrations. In contrast, the glycine-rich C-terminal domain (mEGFP-AtGRP791-176) exhibited rapid condensation kinetics under molecular crowding at protein concentrations much lower than that of the full-length protein. FRAP experiments with mEGFP-AtGRP791-176 droplets provided evidence of their liquid-like behavior, showing rapid and complete fluorescence recovery. Additionally, we investigated the impact of deleting 8 Tyr residues at the C-terminal tail in AtGRP7 LLPS. Consequently, the phase separation propensity of mEGFP-AtGRP7Δ8Tyr was considerably lower compared to mEGFP-AtGRP71-176, suggesting that the C-terminal glycine-rich disordered tail drives AtGRP7 LLPS.