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

摘要 DDX3X是一种X连锁的RNA解旋酶，它的从头突变占女性智力障碍(ID)的1-3% 并与多种表型有关，包括发育迟缓、癫痫、自闭症和大脑 畸形。大约一半的突变是无稽之谈，而另外50%是错误的 突变。初步报告表明，DDX3X突变主要导致功能丧失表型。 然而，我们的合作者在DDX3X个体中发现了反复出现的错义突变，这些突变一直是 与更严重的临床结局和其他DDX3X中未观察到的特定脑畸形有关 个人。这些数据有力地表明，一些DDX3X错义突变具有显性负性表型。 我们进一步发现，DDX3X是大脑正常发育所必需的；具体地说，DDX3X在 胚胎小鼠的大脑会削弱神经前体细胞制造神经元的能力。我们还展示了 与严重损害相关的DDX3X错义突变几乎完全丧失解旋酶活性， 损伤关键靶点的翻译，形成异位的RNA-蛋白质颗粒。这些发现表明，异位 RNA-蛋白质颗粒和异常的DDX3X蛋白质相互作用可能有助于疾病的严重程度。然而， 这些机制还没有得到检验。在这项建议中，我们试图阐明其分子发病机制。 DDX3X错义突变与DDX3X综合征严重的临床缺陷有关。我们将检验这一假设 临床上严重的DDX3X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DDX3X颗粒在神经前体和神经元中表达的特征 轻微和严重的DDX3X错义突变。我们还将确定颗粒是否与受损有关 神经发生。其次，我们将在神经前体和神经元中使用无偏见的蛋白质组筛选来确定 不同的DDX3X错义突变如何损害WT蛋白相互作用组。这些研究对于 了解与DDX3X相关的显性负性表型的分子基础 综合症。此外，我们的研究将广泛地建立一个理解其他神经发育的范例 核糖核酸调控起中心作用的紊乱。

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.