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。DHX和DDX解旋酶家族几个成员的基因编码突变最近与神经发育障碍（ndd）有关。我们最近将DHX30添加到这个不断增长的疾病基因列表中，通过鉴定一组携带杂合的患者，新生错义突变影响编码蛋白的关键残基。我们最初的功能研究揭示了RNA结合或ATP水解的缺陷；在细胞水平上，这与压力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是应力颗粒形成所必需的；此外，通过蔗糖梯度分析发现，DHX30表达缺失导致80S单体峰降低，这清楚地说明了DHX30在翻译调控中的作用。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，我们计划解决两个主要的，高度相关的问题：(1)什么是DHX30的细胞功能，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影响其翻译速率的mrna，并研究失去DHX30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，我们将分析DHX30与另一种ndd相关的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（如树突分支和突触形成）。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。这些将分化成神经元，使我们能够分析DHX30突变对神经元蛋白质合成的影响。此外，在患者来源的神经元中，我们将研究形态学和突触形成。这些研究将通过对携带特定患者突变的小鼠的分析来支持，因为后一种实验系统除了前面提到的生化和形态学研究外，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，并进一步描述翻译在早期神经发育中的作用。