Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly

Removal of introns from pre-mRNA is an essential step of gene expression. T he splicing reaction is catalyzed in a large complex termed the spliceosome . Introns are recognized during the early steps of spliceosome assembly wit h the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is es sential in yeast, also interacts with the U2AF(65)/Mud2 splicing factor. Ho wever, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present t ogether with Mud2 in commitment complex 2 (CC2), but is not detectable in c ommitment complex 1 (CC1). Furthermore, genetic and biochemical depletion o f BBP demonstrated that it is required for CC2 formation. In addition we ob served that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released durin g or immediately after pre-spliceosome formation. interestingly depletion o f BBP or disruption of MUD2 had no significant effect on pre-spliceosome fo rmation and splicing in vitro but led to a transient accumulation of CC1. T hese observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.