The RNA helicase DHX30: Physiological function and role in a neurodevelopmental disorder

依赖于ATP的RNA解旋酶参与了RNA二级结构和RNP颗粒的重塑。最近，DHX和DDX解旋酶家族中几个成员的基因编码突变与神经发育障碍(NDDS)有关。我们最近通过鉴定一组携带杂合的、影响编码蛋白关键残基的从头开始的错义突变的患者，将DHX30添加到这个不断增长的疾病基因列表中。我们最初的功能研究显示，在RNA结合或ATP水解方面存在缺陷；在细胞水平上，这与应激颗粒的形成和蛋白质合成的停止有关。在最近的工作中，我们观察到DHX30是形成应激颗粒所必需的；此外，观察到DHX30的表达缺失导致蔗糖梯度分析的80s单体峰减少，这清楚地表明DHX30在翻译调节中的作用。重要的是，DHX30在中枢神经系统中的功能相关性完全不清楚。在这里，我们计划解决两个高度相关的主要问题：(1)DHX30的细胞功能是什么，特别是在神经元中；(2)患者来源的DHX30突变如何影响中枢神经系统中这种RNA解旋酶的分子和细胞功能，并导致严重的神经发育表型？由于我们的初步数据表明DHX30在控制翻译中的作用，我们将寻找受DHX30影响翻译速率的mRNAs，并研究DHX30的丢失如何改变细胞蛋白质组。我们将分析DHX30与导致应激颗粒形成的信号通路的相关性。此外，我们还将分析DHX30的S与另一种新城疫相关的RNA解旋酶DDX3X相互作用的功能结果。在原代培养的神经元中，我们将分析DHX30的缺失如何影响神经元蛋白质的合成、形成或树突mRNA颗粒，以及特定的神经元参数，如树突分支和突触形成。为了确定患者来源的突变如何干扰DHX30的神经功能，我们将从患者的成纤维细胞中产生诱导的多能干细胞。这些细胞将被分化为iNeurons，使我们能够分析DHX30突变对神经元蛋白质合成的影响。此外，在患者来源的iNeurons中，我们将研究形态和突触形成。这些研究将得到对携带选定患者突变的小鼠的分析的支持，因为后一种实验系统除了前面提到的生化和形态研究外，还允许进行行为研究。综上所述，我们的项目旨在揭示DHX30相关神经发育障碍的分子机制，并进一步阐明翻译在早期神经发育中的作用。

ATP-dependent RNA helicases are involved in the remodeling of RNA secondary structures and RNP particles. Mutations in genes coding for several members of the DHX and DDX helicase families have recently been associated with neurodevelopmental disorders (NDDs). We have recently added DHX30 to this growing list of disease genes by identification of a cohort of patients bearing heterozygous, de novo missense mutations affecting critical residues of the encoded protein. Our initial functional studies revealed deficits in either RNA binding or ATP hydrolysis; on a cellular level, this was associated with formation of stress granules and a shutdown of protein synthesis. In recent work, we observed that DHX30 is required for stress granule formation; in addition, the observation that loss of DHX30 expression causes a reduction in the 80S monosome peak upon sucrose gradient analysis clearly points to a role of DHX30 in the regulation of translation. Importantly, the functional relevance of DHX30 in the central nervous system is completely unclear. Here we plan to address two major, highly interrelated questions: (1) what is the cellular function of DHX30, particularly in neurons; and (2) how do patient derived mutations in DHX30 affect the molecular and cellular functions of this RNA helicase in the central nervous system, and lead to a severe neurodevelopmental phenotype? As our initial data point to a role of DHX30 in controlling translation, we will search for mRNAs that are affected in their translational rate by DHX30 and investigate how the cellular proteome is altered by loss of DHX30. We will analyze the relevance of DHX30 for signaling pathways leading to stress granule formation. Additionally, we will analyze the functional consequence of the DHX30´s interaction with DDX3X, another NDD-related RNA helicase.. In primary cultured neurons, we will analyze how loss of DHX30 affects neuronal protein synthesis, formation or dendritic mRNA granules, and specific neuronal parameters such as dendrite branching and synapse formation. To determine how patient derived mutations interfere with the neuronal function of DHX30, we will generate induced pluripotent stem cells from patient’s fibroblasts. These will be differentiated into iNeurons, allowing us to analyze the effects of DHX30 mutations on neuronal protein synthesis. Furthermore, in patient derived iNeurons we will study morphology and synapse formation. These studies will be backed up by the analysis of mice carrying selected patient mutations, as this latter experimental system also allows for behavioural studies, in addition to biochemical and morphological studies mentioned before. Taken together, our project aims to unravel the molecular mechanisms involved in the DHX30-associated neurodevelopmental disorder and further delineate the role of translation in early neurodevelopment.