Abstract
The spliceosome is the complex macromolecular machine responsible for removing introns from pre-mRNAs. The processes of spliceosome assembly and activation rely on the coordinated interactions of many dozens of spliceosome components to identify splice sites in a pre-mRNA, build a spliceosome, and activate the spliceosome for catalysis by formation of an active site prior to transesterification. The activation step itself likely involves many intermediates. It results in loss of the U1 and U4 snRNPs from the spliceosome, removal of SF3 from the branchsite, and several conformational rearrangements of the snRNAs and pre-mRNA prior to lariat formation. These assembly and activation events are best studied using endogenous spliceosome components found in whole or nuclear cell extracts. We recently demonstrated that a single molecule technique (CoSMoS: Co-localization Single Molecule Spectroscopy) in combination with yeast genetic engineering and chemical biology provides a powerful method for studying spliceosome assembly in S. cerevisiae whole cell lysate (Hoskins et al., Science, v331, pg. 1289-95 (2011)). That study provided significant novel insight into the kinetics of the assembly reaction. We are now extending these results to spliceosome activation. By monitoring the relative association and dissociation kinetics of the U1, U4, U5, NTC, and SF3b spliceosome components on single pre-mRNAs, we are able to definitively order the snRNP association and dissociation events involved in spliceosome activation.