Using Single Molecules to Map the Pathways of Spliceosome Active Site Formation

Eukaryotic pre-mRNA splicing is carried out by a large macromolecular machine composed of dozens of proteins and 5 small nuclear RNAs called the spliceosome. Spliceosomes are not processive. For removal of each intron, a new spliceosome is assembled, creates a RNA active site, catalyzes splicing, and is disassembled. Cryo-EM structures have recently elucidated a number of stable intermediates formed during these steps. However, we have little knowledge of how transitions between these intermediates occur or on the composition of transiently formed complexes. We have used colocalization single molecule spectroscopy (CoSMoS) to follow the formation of the spliceosome active site (activation) in real time. From the single molecule data, we have elucidated the kinetic scheme for spliceosome activation and identified several novel, transiently formed intermediate complexes. These results complement recent advances in spliceosome biochemistry by cryo-EM and reveal novel targets for future structural elucidation.