Isolation of an active step I spliceosome and composition of its RNP core

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome’s active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19–CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19–CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome’s catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure. The splicing process, whereby unwanted introns are clipped out to produce a definitive functional RNA, occurs in a large RNA/protein complex called the spliceosome. Its complexity has hampered efforts to determine what the core components necessary for splicing are. A prerequisite for addressing this question is the purification of spliceosomal complexes that are active even in the absence of added factors. To this end, Bessonov et al. have purified two intermediary complexes of the splicing reaction. The differences between these complexes provide an important and long-awaited glimpse into the composition of the catalytic core. Splicing is carried out within a large structure, the spliceosome, containing many RNAs and proteins. In this paper, two intermediary complexes of the splicing reaction have been purified. The differences in these complexes provide a significant advancement in understanding of the composition of the catalytic core.