Cell biological and proteomic investigation of pathogenic DDX3X missense mutations during neurogenesis

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Abstract De novo mutations in DDX3X, an X-linked RNA helicase, account for 1-3% of intellectual disability (ID) in females and are associated with a broad range of phenotypes, including developmental delay, epilepsy, autism and brain malformations. Approximately half of the mutations are nonsense, whereas the other 50% are missense mutations. Initial reports suggested that DDX3X mutations primarily cause loss-of-function phenotypes. However, our collaborators identified recurrent missense mutations in DDX3X individuals that were consistently associated with more severe clinical outcomes and specific brain malformations not observed in other DDX3X individuals. These data strongly suggest some DDX3X missense mutations exert dominant negative phenotypes. We have further discovered that DDX3X is required for proper brain development; specifically, loss of Ddx3x in the embryonic mouse brain impairs the ability of neural progenitors to make neurons. We also demonstrated that DDX3X missense mutations associated with severe impairment have nearly complete loss of helicase activity, impaired translation of key targets, and form ectopic RNA-protein granules. These findings indicate that ectopic RNA-protein granules and aberrant DDX3X protein interactions may contribute to disease severity. However, these mechanisms have not been examined. In this proposal, we seek to elucidate the molecular pathogenesis of DDX3X missense mutations linked to severe clinical deficits in DDX3X syndrome. We will test the hypothesis that clinically severe DDX3X missense mutations perturb its cellular dynamics and protein interactome during neurogenesis. First, we will characterize DDX3X granules in neural progenitors and neurons expressing mild and severe DDX3X missense mutations. We will also determine whether granules are linked to impaired neurogenesis. Second, we will use an unbiased proteomic screen in neural progenitors and neurons to determine how different DDX3X missense mutations impair the WT protein interactome. These studies are critical for understanding the molecular underpinnings of dominant negative phenotypes associated with DDX3X syndrome. Further, our studies will broadly establish a paradigm for understanding other neurodevelopmental disorders in which RNA regulation plays a central role.