Splicing of ultraconserved poison exons controls mitotic fidelity and stem cell viability

SR proteins are essential splicing regulators whose expression is controlled in part through poison exons (PEs) - ultraconserved non-coding exons that trigger nonsense-mediated decay - yet the biological functions of these elements remain undefined. Here, we show that homozygous deletion of SRSF3-PE or TRA2β-PE is selected against in mouse embryos and human induced pluripotent stem cells (iPSCs), and that conditional PE deletion causes apoptotic death in iPSCs but is tolerated in post-mitotic neurons, revealing a proliferative-state-specific requirement. Mechanistically, PE deletion elevates SR protein levels, triggers widespread splicing dysregulation, and disrupts the correct splicing of a mitotic gene network associated with spindle defects and mitotic errors. These findings establish ultraconserved poison exons as essential regulators of mitotic splicing fidelity and stem cell viability.