Poison exon splicing in the human brain: a new frontier for understanding and targeting neurological disorders

Poison exons (PE) are highly conserved exon cassettes whose inclusion creates premature termination codons (PTCs) and triggers nonsense-mediated decay (NMD) of the mature transcript, therefore reducing protein expression. Despite their important role in post-transcriptional regulation, PEs remain poorly annotated due to the lack of systematic, transcriptome-wide approaches. In this study, we comprehensively investigated the function of PEs in the human brain and assessed the impact of pathogenic variants on their splicing. A comparative analysis of 957 eukaryotic transcriptomes revealed that humans exhibit the highest enrichment of NMD-targeted transcripts. To delineate the full landscape of PEs in the human transcriptome, we considered conserved (PhyloP score ≥ 20) alternatively spliced exons, less than 300 base pairs (bp) long, with the ability to introduce PTCs positioned more than 150 base pairs (bp) downstream of the transcription start site and outside the last two exons of the transcript. Our analysis identified 12,014 PEs in the human genome. For each PE, we calculated the percent-spliced-in (PSI) value across tissue types and developmental stages using GTEx and BrainSpan RNA-seq dataset, respectively. Overall, we identified 117 PEs uniquely found in the human brain, 1,214 PEs with brain-specific differential splicing compared to other tissues, and 1,610 PEs which splicing change during brain development. Integrating ClinVar and SpliceAI, we identified 1,877 annotated pathogenic variants predicted to affect the splicing of 891 PEs in the human brain. Notably, many of these variants were associated with neurodevelopmental and neurodegenerative disorders such as epilepsy, intellectual disability and frontotemporal dementia. We functionally validated the impact of selected variants using an optimized CRISPR prime-editing workflow in human cell lines. Our findings highlight PEs as pivotal regulators of gene expression in the human brain and establish a foundation for therapeutic strategies targeting PE mis-splicing in neurological diseases.