Overexpression of 2nd step splicing factor mutants
Selection of tandem NAGNAG alternative 3'-splice sites (TASS 3'SS) occurs during the catalytic stage of pre-mRNA splicing, when exon-ligation factors stabilize 3'SS docking within the spliceosome active site. Using cryo-EM structures of human spliceosomes assembled on ß-globin pre-mRNA, we reveal the complete architecture of the active site during exon ligation. A novel stacking interaction with the Prp8 a-finger caps 3'SS docking, providing a structural basis for differential stability of competing 3'SS. Weaker stacking at proximal sites requires stabilization by exon-ligation factors, consistent with predictions from machine-learning. Strikingly, the SDE2 N-terminus runs along the branch helix, projecting into the active site to help pair the 5'SS and 3'SS. Prp18 engages the core and positions the FAM32A C-terminus, which rigidifies the active site to promote proximal 3'SS docking. Prp18 and SDE2 may promote efficient flow through the splicing pathway and engage the spliceosome core in a transcript-specific manner, dependent on the strength of the 5' exon and branch helix stability, consistent with their knockdown causing intron retention . Importantly, structure-guided mutation of specific exon-ligation factors restores canonical 3'SS choice at mutant CFTR and BRCA1 NAGNAG sites , demonstrating therapeutic potential. Our results define how exon-ligation factors and conserved RNA-protein interactions stabilize 3'SS docking, providing a molecular framework for regulated alternative 3'SS choice during catalysis.