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

摘要 DDX 3X（一种X连锁RNA解旋酶）的新生突变占女性智力残疾（ID）的1-3% 并与广泛的表型相关，包括发育迟缓，癫痫，自闭症和脑 畸形大约一半的突变是无义的，而另外50%是错义的。 突变。最初的报告表明，DDX 3X突变主要导致功能丧失表型。 然而，我们的合作者在DDX 3X个体中发现了经常性的错义突变，这些突变与DDX 3X个体的错义突变一致。 与更严重的临床结局和其他DDX 3X中未观察到的特定脑畸形相关 个体这些数据强烈表明，一些DDX 3X错义突变发挥显性负性表型。 我们进一步发现，DDX 3X是大脑正常发育所必需的;特别是， 胚胎小鼠脑损害神经祖细胞产生神经元的能力。我们还证明了 与严重损伤相关的DDX 3X错义突变几乎完全丧失解旋酶活性， 关键靶点的翻译受损，并形成异位RNA-蛋白质颗粒。这些发现表明，异位 RNA-蛋白颗粒和异常DDX 3X蛋白相互作用可能导致疾病严重程度。然而，在这方面， 这些机制尚未得到审查。在这个建议中，我们试图阐明分子发病机制 DDX 3X错义突变与DDX 3X综合征的严重临床缺陷有关。我们将检验这个假设 临床上严重的DDX 3X错义突变扰乱了其细胞动力学和蛋白质相互作用组， 在神经发生过程中。首先，我们将描述DDX 3X颗粒在神经祖细胞和神经元表达中的特征， 轻度和重度DDX 3X错义突变。我们还将确定颗粒是否与受损的 神经发生其次，我们将在神经祖细胞和神经元中使用无偏见的蛋白质组筛选来确定 不同的DDX 3X错义突变如何损害WT蛋白相互作用组。这些研究对于 了解DDX 3X相关显性阴性表型的分子基础 综合征此外，我们的研究将广泛建立一个范式，了解其他神经发育 RNA调节起核心作用的疾病。

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.