Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3 ' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spli-ceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3 ' ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3 ' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3 ' ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3 ' ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3 ' ss choices.