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Abstract

The spliceosome excises introns from pre-mRNA in a two-step splicing reaction. So far, the three-dimensional (3D) structure of a spliceosome with preserved catalytic activity has remained elusive. Here, we determined the 3D structure of the human, catalytically active step I spliceosome (C complex) by cryo-electron microscopy (cryo-EM) in vitrified ice. Via immunolabeling we mapped the position of the 5′ exon. The C complex contains an unusually salt-stable ribonucleoprotein (RNP) core that harbors its catalytic center. We determined the 3D structure of this RNP core and also that of a post-step II particle, the 35S U5 snRNP, which contains most of the C complex core proteins. As C complex domains could be recognized in these structures, their position in the C complex could be determined, thereby allowing the region harboring the spliceosome's catalytic core to be localized.