Eukaryotic pre-mRNA splicing is carried out by a MDa-sized ribonucleoprotein complex called the spliceosome. Spliceosomes must undergo an activation process to assemble and fold the active site used for splicing catalysis. During this process, two snRNAs and around fifty proteins are exchanged into and out of the yeast spliceosome. Many of these factors enter and leave the spliceosome as part of well-defined subcomplexes (e.g., the U1 snRNP, the Prp19-containing complex or NTC) while for others it is unclear if their association or release occurs step-wise or concertedly. Despite available cryo-EM structures of spliceosomes before and after activation, it remains unclear how these large-scale rearrangements occur, and the molecular compositions of transiently formed, on-pathway activation intermediates are unknown. We have used Colocalization Single Molecule Spectroscopy (CoSMoS) to observe the recruitment and release of splicing factors (Lsm2-8, Snu66, Snu23, Prp38, and Spp381) to spliceosomes during activation. We have characterized their binding kinetics under conditions which both permit and inhibit activation and relative to release and recruitment of the U4 snRNP and NTC.  The B complex-specific proteins (Snu23, Prp38, and Spp381) all exhibit similar binding and dissociation kinetics from spliceosomes and are distinct from Snu66. This suggests that the B complex-specific proteins function as a single unit during assembly and activation. Further, B complex-specific proteins do not join the spliceosome concomitantly with the tri-snRNP, rather they are integrated ~58 seconds after tri-snRNP binding. The spliceosome complex containing both the U4 snRNP and B-complex specific proteins persists for ~10 seconds before U4 snRNP destabilization which is then followed ~60 seconds later by release of the B-complex specific proteins.  The Lsm2-8 complex is released late in activation and after U4 snRNP destabilization and recruitment of the NTC. Spliceosome complexes containing both the Lsm2-8 ring and NTC are also transient with lifetimes of ~30 seconds. In sum, our results allow us to construct a kinetic map of the order of events occurring during activation as well as define the molecular composition of several novel, transient activation intermediates not yet captured by cryo-EM.<br/>

Mapping the pathway for creation of the spliceosome active site with single molecule fluorescence microscopy

ACS Fall 2020 Virtual Meeting

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