The role of RNA binding proteins in neurodevelopmental disorders

Abstract

Neurodevelopmental disorders (NDDs) represent a large group of disorders arising from alterations of tightly coordinated processes that regulate development and function of the brain. Different types of syndromes, including intellectual disability, autism spectrum disorders, attention deficit hyperactivity disorders and epilepsy, have been classified as NDDs (Niemi et al., 2018). Due to their relatively high prevalence, NDDs constitute a serious socio-economic problem of health care. The identification of NDD-associated genes is essential for a better understanding of both the genetic contribution to the disease and the potential underlying pathomechanism. The recent introduction of high-throughput technologies such as next generation sequencing (NGS) has substantially improved our understanding of the genetic causes of different forms of NDDs (Vissers et al., 2016). Specifically, NGS-based studies in children affected by NDDs have led to the identification of mutations in genes encoding proteins involved in RNA metabolism as a major cause of NDDs; this includes AGO2, a component of the RNA interference pathway, as well as several RNA helicases such as DHX30. The DHX30 gene was initially established as a candidate gene for NDDs (Eldomery et al., 2017; Zheng et al., 2015). The identification of mutations in AGO2 in 21 patients, and in DHX30 in more than 35 patients affected by mild to severe NDDs has ultimately provided strong evidence for assessing the indispensability of these genes in the development of the central nervous system. For DHX30, the majority of the affected individuals carry heterozygous missense variants within highly conserved helicase core motifs (HCMs) and display global developmental delay, intellectual disability, severe speech impairment and gait abnormalities. Notably, four individuals carrying heterozygous variants which result in haploinsufficiency or truncated proteins present with a milder clinical course, thereby delineating a different, less severe clinical subtype compared to variants affecting the HCMs. However, the physiological role of DHX30 in cellular RNA metabolism so far remains largely unexplored. The current PhD project was focused on analyzing the molecular and cellular dysfunction associated with AGO2 and, as a central topic, DHX30 missense mutations and on elucidating the physiological role of DHX30 in cellular RNA metabolism. In ATPase assays, it is shown here that DHX30 is an RNA-dependent ATPase, and that all DHX30 missense mutations affecting highly conserved HCMs lead to disrupted ATPase activity. Using purified recombinant protein in helicase assays with a radiolabeled RNA duplex, DHX30 is formally established here as an ATP-dependent RNA helicase. Again, all tested missense mutations in HCMs disrupt the helicase activity whereas a mutation outside the core region does not affect helicase activity. Subcellular localization studies confirmed the cytoplasmic diffuse distribution of overexpressed GFP-tagged DHX30-wild type (WT). All HCM missense variants, overexpressed as GFP-fusion proteins, stimulated spontaneous stress granule (SG) hyper-assembly, leading to inhibition of global translation. Again, mutations outside HCMs had lower to no ability to induce SGs. The clear correlation between SG formation and severity of the phenotype observed in the patients motivated further investigation of the role of DHX30 in SG assembly. Permanent knockdown using CRISPR/Cas9-based gene editing of DHX30 in a human cell line led to impaired SG formation upon heat stress, uncovering a previously unknown role for DHX30 in this aspect of RNA metabolism. Analysis of the nature of DHX30 missense mutations showed that RFP-tagged DHX30 WT is recruited to SGs upon co-expression of several GFP-tagged missense variants, possibly suggesting a dominant negative effect of the mutations. However, the observation that both endogenous and overexpressed DHX30-WT are recruited to SGs after stress induction (Lessel et al., 2017) together with the fact that DHX30-deficient cells show reduced SG formation, actually suggest that mutations affecting HCMs result in a detrimental gain of function with respect to SG formation.