CATION-π INTERACTIONS BETWEEN Arg AND Tyr PAIRS AT THE DISORDERED C-TERMINAL TAIL GOVERN AtGRP7 PHASE SEPARATION

The clock-regulated RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich protein 7) is pivotal in regulating key physiological processes in A. thaliana, such as circadian rhythm, flowering time, and abiotic stress responses. AtGRP7 consists of an N-terminal RNA recognition motif (RRM) domain and a C-terminal disordered tail composed of glycine-rich repeats. Here, we investigated AtGRP7’s ability to undergo liquid-liquid phase separation (LLPS), a phenomenon underlying the formation of membraneless organelles, using full-length protein and mutants with isolated domains to elucidate the role of RNA binding. We expressed various AtGRP7 constructs (full-length, N-terminal, C-terminal domains, Δ8Tyr, and Δ8Arg) fused with mEGFP in Escherichia coli BL21 DE3 and purified them through nickel-affinity and size exclusion chromatography. LLPS was assessed via fluorescence microscopy and turbidity assays. Under molecular crowding conditions, AtGRP7-FL formed spherical, micrometer-sized condensates. These condensates displayed high sensitivity to NaCl but partial resistance to the aliphatic alcohol 1,6-hexanediol, indicating a significant role of polar interactions in AtGRP7 LLPS. The addition of a specific RNA ligand (32 nucleotides) disrupted AtGRP7 condensation in a dose-dependent manner, suggesting that RNA binding maintains AtGRP7 in the diffuse state. The isolated N-terminal domain (AtGRP7-NTD) did not undergo LLPS, even at elevated protein concentrations. Conversely, the glycine-rich C-terminal domain (AtGRP7-CTD) exhibited rapid condensation kinetics at much lower protein concentrations compared to AtGRP7-FL, as demonstrated by Csat experiments. Fluorescence recovery after photobleaching (FRAP) experiments on AtGRP7-FL and AtGRP7-CTD droplets confirmed their liquid-like properties, with approximately 30% fluorescence recovery within 200 seconds. Moreover, we examined the impact of deleting 8 Tyr or 8 Arg residues in the C-terminal tail on AtGRP7 LLPS. These constructs did not undergo LLPS, suggesting that these residues function as "stickers" forming cation-π interactions critical for AtGRP7 LLPS.