Phase Separation Driven by Dynamic Interactions in the N-Terminal Intrinsically Disordered Region of the DEAD-Box RNA Helicase DDX3X

The formation of membraneless organelles via liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as a central mechanism of cellular compartmentalization to finely regulate biological processes. DDX3X, a member of the DEAD-box RNA helicase family, is one of the global regulators of RNA-containing phase-separated organelles. While the importance of DDX3X in organelle formation is well-recognized, the molecular mechanisms underlying its RNA-driven LLPS remain poorly understood. In this study, we focused on the dynamic interactions between the N-terminal intrinsically disordered region (N-IDR) of DDX3X and G-quadruplex (GQ) RNA, which is a key regulator of physiological membraneless organelle assembly owing to its unique ability to promote LLPS. Using solution nuclear magnetic resonance spectroscopy, we identified hotspot regions for self-assembly within the N-IDR. These regions comprise charged stretches interspersed with key aromatic residues, whose interactions drive LLPS through a combination of electrostatic and π-interactions. Binding of GQ RNA effectively strengthens intermolecular interactions involving the arginine-rich segments of the N-IDR, providing molecular insights into its RNA-driven LLPS. We further discuss the functional implications of GQ-specific granule formation under stress conditions, highlighting the potential roles of DDX3X-GQ RNA interplay in cellular translational regulation.