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

RNA 解旋酶 DHX30：神经发育障碍中的生理功能和作用

• 批准号: 463129991
• 负责人: Professor Dr. Hans-Jürgen Kreienkamp
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/463129991?language=en
• 关键词: RNA; helicase; DHX30; Physiological; function

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.

ATP 依赖性 RNA 解旋酶参与 RNA 二级结构和 RNP 颗粒的重塑。编码 DHX 和 DDX 解旋酶家族几个成员的基因突变最近与神经发育障碍 (NDD) 相关。通过鉴定一组携带影响编码蛋白关键残基的杂合、从头错义突变的患者，我们发现了越来越多的疾病基因列表。 ATP 水解；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。蔗糖梯度分析中 80S 单体峰的减少清楚地表明 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是完全相关的。在这里，我们计划解决两个主要的、高度相关的问题：(1) DHX30 的细胞功能是什么，特别是在神经元中；(2) DHX30 中患者衍生的突变如何影响这种 RNA 解旋酶的分子和细胞功能。当我们的初始数据表明 DHX30 在控制翻译中的作用时，我们将寻找受 DHX30 影响的 mRNA 并研究其如何影响中枢神经系统，并导致严重的神经发育表型？ DHX30 的丢失会改变细胞蛋白质组。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，我们将分析 DHX30 与另一种 NDD 相关 RNA 解旋酶 DDX3X 相互作用的功能后果。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合成、树突 mRNA 颗粒的形成以及特定神经元参数（例如树突）为了确定患者衍生的突变如何干扰 DHX30 的神经元功能，我们将从患者的成纤维细胞中产生诱导多能干细胞，这些细胞将分化为 iNeurons，从而使我们能够分析 DHX30 突变对神经元蛋白质合成的影响。此外，在源自患者的 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，就像后一个实验系统一样。除了前面提到的生化和形态学研究之外，还可以进行行为研究。总之，我们的项目旨在揭示 DHX30 相关神经发育障碍的分子机制，并进一步描述翻译在早期神经发育中的作用。