PROBING DYNAMIC PROPERTIES OF DDX3X CONDENSATES USING X RAY PHOTON CORRELATION SPECTROSCOPY

DDX3X is a member of the DEAD box helicase family largely associated with neurodevelopment and neurodevelopmental diseases (NDD). Importantly, DDX3X promotes liquid-liquid phase separation (LLPS) in stress granules (SG). In this study, we characterized the dynamics of DDX3X liquid condensation in the absence and in the presence of RNA using X ray photon correlation spectroscopy (XPCS) at Sirius. DDX3X droplet formation was also followed over time by fluorescence microscopy. The relaxation dynamics of DDX3X condensates was characterized by a diffusive, Brownian motion with the Kohlrausch-Williams-Watts decay function exponent γ around 1 and a relaxation time ranging from 0.25 to 0.90 seconds during the incubation time over the entire dynamic size scale. As the condensates matured during incubation, we observed a slight shift towards a superdiffusive (γ > 1) viscoelastic relaxation, suggesting network fluctuations within the condensates. No major alteration in the XPCS Kohlrausch-Williams-Watts decay function exponent was observed in the presence of either single-stranded (ss-) or double-stranded (ds-) RNA molecules at 1:0.5 (protein:RNA) stoichiometry, indicating that RNA molecules do not significantly alter the material properties of DDX3X droplets over time under these conditions. However, both ssRNA and dsRNA impaired liquid-liquid phase separation at high concentrations. Furthermore, NDD-linked mutation in the RNA-binding region of DDX3X resulted in a liquid-to-solid transition upon droplet maturation over time. Together, our results provide new insights into the molecular mechanisms of DDX3X condensation and the molecular pathology of a severe NDD-associated mutant.