Pigini, P. | bioRxiv Science

bioRxiv2025

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

BackgroundPoison exons (PE) are highly conserved exons whose inclusion creates premature termination codons (PTCs) and triggers nonsense-mediated decay (NMD) of the mature transcript. Despite their important role in post-transcriptional regulation, PEs remain poorly annotated due to the lack of systematic transcriptome-wide approaches. ResultsA comparative analysis of 957 eukaryotic transcriptomes revealed that Homo Sapiens exhibits the highest enrichment of NMD-targeted isoforms. By systematically predicting and annotating splicing events that generate PTCs, we found 9,814 PEs in the human genome. Using RNA-seq dataset from GTEx and BrainSpan, we analyzed PE splicing across tissues and developmental stages and identified 83 PEs uniquely found in the human brain, 973 PEs with brain-specific differential splicing, and 1,284 PEs differentially spliced during brain development. By integrating ClinVar variant annotations with SpliceAI predictions, we identified 1,625 pathogenic variants associated with neurological disorders that are predicted to affect the splicing of 743 PEs, and we functionally validated the impact of a subset of them using CRISPR prime-editing in human cells. Functional characterization of the deep intronic NDUFAF6 c.420+784C>T variant associated with Leigh syndrome demonstrated increased PE inclusion and a significant reduction in NDUFAF6 protein levels in iPSC-derived induced neurons. Mutant cells also exhibited impaired mitochondrial membrane potential compared to controls. Together, these findings provide functional evidence that the c.420+784C>T variant promotes aberrant PE inclusion, leading to reduced NDUFAF6 expression and mitochondrial dysfunction. ConclusionsOur findings highlight PEs as pivotal regulators of gene expression in the human brain and support the therapeutic targeting of PE splicing in neurological diseases.

genetics↗

bioRxiv2025

MAPT Splicing Modulators as a Therapeutic Strategy for Tauopathies

Tauopathies are neurodegenerative diseases characterized by the abnormal accumulation of microtubule-associated protein tau (MAPT) in the brain. These disorders, like frontotemporal dementia (FTD-Tau), currently lack effective therapies and can occur sporadically or be inherited when associated with MAPT gene mutations. The MAPT gene region encompassing exon 10 and adjacent introns is a hotspot for pathogenic variants, including splicing mutations that enhance exon 10 inclusion and increase 4R tau expression, and gain-of-function mutations that generate aggregation-prone mutant 4R tau protein. For these 4R-specific tauopathies, a targeted mRNA splicing approach that promotes exon 10 exclusion may offer therapeutic benefit. In this study, we discovered novel splicing modulator compounds (SMCs) that promote MAPT exon 10 exclusion, and demonstrated their efficacy in FTD patient-derived neuronal models carrying the tau-P301L gain-of-function mutation or the tau-S305N splicing mutation. Treatment with SMC reduced 4R tau expression and decreased the accumulation of hyperphosphorylated tau (pTau), oligomeric and insoluble tau, thereby rescuing tau-associated neuronal toxicity. Importantly, our lead SMC corrected the 3R/4R splice ratio in vivo and significantly reduced pTau in the brain of a gene- replacement (GR) mouse model expressing the human tau-N279K splicing mutation. These findings support the therapeutic potential of this class of small molecules and establish MAPT pre- mRNA splicing modulation as a promising strategy for the treatment of 4R tauopathies. One Sentence SummaryDiscovery of SMCs that correct MAPT splicing, reduce 4R tau, and rescue pathology in patient- derived neuronal and in vivo models of 4R tauopathies.

neuroscience↗