Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs); however, the role of HNRNPU in human neural development and NDDs remains to be studied. Here, we describe the molecular and cellular outcomes of HNRNPU deficiency during in vitro neural differentiation of isogenic and patient-derived neuroepithelial stem cells. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show the effects of the molecular reorganization at the cellular level, where HNRNPU downregulation leads to altered neurogenesis and an increased fraction of neural progenitors marked in the maturing neuronal population. We conclude that HNRNPU deficiency is responsible for the delayed commitment of neural progenitors to neuronal maturation, ultimately leading to altered neurogenesis.

Autism spectrum disorder (ASD) is a heterogenous, early-onset neurodevelopmental disorder with multifactorial aetiology. There are hundreds of genes that contribute to ASD. Additionally, several environmental factors, such as neurotoxic compounds, endocrine modulators, and neuropsychiatric medications, have been associated with ASD outcomes. We investigated a highly efficient and multiplexable, fractional factorial experimental design to study multiple environmental factors and human induced pluripotent stem cell (iPSC) lines in the context of ASD. RNA expression analysis revealed significant upregulation of pathways related to synaptic function and lipid metabolism, following exposure to lead and fluoxetine, respectively. We validated our findings using mass spectrometry-based metabolomics after fluoxetine exposure. The study demonstrates that the fractional factorial experiment design can be used for multiplexed transcriptomic analyses to detect relevant changes at the pathway level. Our study provides a roadmap for understanding the impact of low-grade environmental exposures on human neurodevelopment. Expansive future studies will require multiple cell lines with different genetic backgrounds for characterising environmental exposures in ASD and other neurodevelopmental disorders.

Background Circular RNA (circRNA) molecules, generated through non-canonical back-splicing of exon-exon junctions, have recently been implicated in diverse biological functions including transcriptional regulation and modulation of protein interactions. CircRNAs are emerging as a key component of the complex neural transcriptome implicated in brain development. However, the specific expression patterns and functions of circRNAs in human neuronal differentiation have not been explored. Results Using total RNA sequencing analysis, we identified expressed circRNAs during the differentiation of human neuroepithelial stem (NES) cells into developing neurons and discovered that many circRNAs originated from host genes associated with synaptic function. Interestingly, when assessing population data, exons giving rise to circRNAs in our dataset had a higher frequency of genetic variants. Additionally, screening for RNA-binding protein sites identified enrichment of Splicing Factor Proline and Glutamine Rich (SFPQ) motifs in increased circRNAs, several of which were reduced by SFPQ knockdown and enriched in SFPQ ribonucleoprotein complexes. Conclusions Our study provides an in-depth characterisation of circRNAs in a human neuronal differentiation model and highlights SFPQ as both a regulator and binding partner of circRNAs elevated during neuronal maturation.