Abstract
Spliceosome assembly and catalytic activation involve dozens of protein and snRNA binding and unbinding events. The B-complex specific proteins (Prp38, Snu23, and Spp381) have critical roles in stabilizing the spliceosome during rearrangements essential for activation. While these proteins are conserved, different mechanisms have been proposed for their recruitment to spliceosomes. To visualize recruitment directly, we used Colocalization Single Molecule Spectroscopy (CoSMoS) to study the dynamics of Prp38, Snu23, and Spp381 during splicing in real time. These proteins can bind to and release from spliceosomes simultaneously and are likely associated with one another. We designate the assembly minimally containing Prp38, Snu23, and Spp381 as a potential B complex protein (BCP) subcomplex of the spliceosome. Under splicing conditions, BCP proteins associate with pre-mRNA after tri-snRNP binding. BCP protein release predominantly occurs after U4 snRNP dissociation and after NineTeen Complex (NTC) association. Under low concentrations of ATP, BCP proteins preassociate with the tri-snRNP resulting in their simultaneous binding to pre-mRNA. Together, our results reveal that BCP protein recruitment to the spliceosome is conserved between Saccharomyces cerevisiae and humans. The binding of BCP proteins to the tri-snRNP when ATP is limiting may result in the formation of unproductive complexes that could be used to regulate splicing.