Abstract
Splicing is a tightly regulated process essential for gene expression and proper cellular function. It involves the removal of introns from precursor messenger RNA (pre-mRNA) to produce mature mRNA. This is mediated by the spliceosome, a dynamic complex composed of over 100 proteins and RNA molecules. Although splicing occurs in all cells, mutations in spliceosomal components can result in tissue-specific developmental disorders, particularly affecting the brain. The mechanisms behind this tissue specificity remain poorly understood.
We identified nine unrelated individuals from around the world, including one from New Zealand, with non-inherited (de novo) missense variants affecting the conserved amino acid arginine at position 267 (Arg267) in the spliceosomal gene CRNKL1 (Crooked Neck Pre-mRNA Splicing Factor 1), a key regulator of spliceosome activation. All individuals shared a strikingly similar phenotype, including microcephaly, epilepsy, and profound developmental delay. We aimed to investigate the pathogenic consequences of this variant using zebrafish embryos as an in vivo model.
To do this, we microinjected zebrafish embryos at the one-cell stage with mRNA encoding either wild-type crnkl1 or the p.Arg105 variant (the zebrafish equivalent of human Arg267). Each condition included 60 embryos and was repeated in triplicate.
Development was assessed at 24 hours post-fertilisation using bright-field microscopy. Results showed that 85–90% of crnkl1 mutant embryos exhibited significant neurodevelopmental abnormalities, including absent brain and eye structures and disrupted body axis elongation. Quantitative analysis revealed a marked developmental delay, with most mutants arrested at the 8–14 somite stage, compared to the Prime-5 stage in controls. Mutant embryos also showed significantly reduced body length (1.86 ± 0.14 mm vs. 2.43 ± 0.17 mm; P<0.0001, unpaired t-test), decreased cell proliferation (P<0.0001, unpaired t-test), and increased cellular stress. Transcriptomic analysis confirmed dysregulation of neuronal and cell cycle pathways.
These findings identify CRNKL1 as a novel disease gene critical for early brain development, highlighting the vulnerability of neurodevelopment to impaired splicing regulation.