Structural basis for alternative 3′ splice site selection in the human spliceosome active center

Accurate alternative splicing requires discrimination between adjacent 3′ splice sites (3′-ss) during catalysis and is disrupted by pathogenic AG-gain mutations that create competing 3′-ss. Here, we present cryo-EM structures of human spliceosomes assembled on native-sequence pre-mRNAs, revealing how the catalytic core controls alternative 3′-ss selection. SDE2 is a previously unrecognized active-center component that promotes a docking-competent spliceosome conformation. Machine learning, in vivo transcriptomics, and in vitro biochemistry show how SDE2 cooperates with FAM32A and Prp18 to act as readers of a cis-regulatory code that governs 3′-ss selection during catalysis. These factors promote weaker, proximal site use by counteracting an intrinsic distal bias generated by active-site interactions with the distal-site -4 nucleotide. Structural or genetic perturbation of these exon-ligation factors destabilizes proximal 3′-ss docking and restores canonical splicing in disease-relevant CFTR and BRCA1 AG-gain alleles. Our work establishes the spliceosome active center as a tunable regulatory hub for alternative splicing.