MOLECULAR MECHANISMS UNDERLYING A CLINICALLY SEVERE DDX3X MISSENSE MUTATION ASSOCIATED WITH INTELLECTUAL DISABILITY

DDX3X is a DEAD-box RNA helicase abundant in the developing brain that has been implicated in the regulation of RNA metabolism. This protein undergoes liquid-liquid phase separation (LLPS) in vitro and it is a component of cellular stress granules (SG), membrane-less organelles (MLOs) where specific RNA molecules and transcripts are sequestered upon cellular stress. Intellectual disability (ID)-associated mutations in DDX3X compromise cell function. We investigated the impact of a DDX3X missense mutation, R326H, identified in patients with severe ID and brain malformation. Over-expression of DDX3X R326H mutant in SH-SY5Y cells resulted in aberrant, 1,6-hexanediol-resistant SG formation in the presence of sodium arsenite. Differently from previously described for other DDX3X ID-linked mutations, our biophysical experiments showed that the R326H mutation does not result in misfolding followed by aggregation of DDX3X in vitro. Our biochemical and structural studies suggest that the R326H mutation triggers structural changes in the DDX3X RNA-binding region impairing RNA interaction and ATPase activity. We are now investigating the role of RNA and ATP in the process of full-length DDX3X R326H condensation in vitro to explain the aberrant LLPS observed in cells. Together, our data aim to deepen the understanding of a crucial function of DDX3X in SG dynamics, demonstrating how the R326H mutation in DDX3X causes SG dysregulation leading to a severe form of intellectual disability.