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

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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.