Towards Three-Color Live-Cell Imaging of Spliceosome Assembly

The process whereby introns are removed from pre-mRNAs occurs in a complex macromolecular machine known as the spliceosome. The spliceosome is dynamic, assembling each of its five small ribonucleoprotein particles (snRNPs) and hundreds of associated proteins prior to each splicing event. This complexity has made characterization of splicing incredibly challenging. While extensive biochemical and biophysical studies have been carried out to elucidate the binding mechanism and dynamics, these studies do not take into consideration the effect of the cellular environment on the kinetics of protein and RNA interactions. We recently demonstrated that fast relaxation imaging (FReI), which combines fluorescence microscopy and temperature jumps, provides a powerful method to study protein-RNA interactions inside a living cell (Guzman Dissertation 2015). That study provided insight into binding kinetics and RNA-mediated localization of the U1A-SL2 complex, components of the U1 snRNP subunit of the spliceosome. We are now developing a three-color FReI that enables the correlation of multiple events and determination of their order and timing. By monitoring the FRET between successively larger complexes, we will be able to determine the association kinetics, dissociation kinetics and coupling within and between snRNP and associated proteins during spliceosome assembly in the native cellular environment.