Single Molecule Evidence for Long Range Interactions During Commitment Complex Formation

Abstract

The spliceosome is a multimegadalton ribonucleoprotein (RNP) consisting of five small nuclear RNPs (snRNPS) that assemble on precursor mRNA (pre-mRNA) in an ordered, dynamic pathway to remove introns. Commitment complex (CC) is the earliest stage of spliceosome assembly and is composed of pre-mRNA, the U1 snRNP, the branchpoint bridging protein (BBP)/mud2 heterodimer, and the cap binding complex (CBC). During CC formation U1 recognizes a short conserved sequence that identifies the 5’ end of the intron known the 5’ splice site (5′SS) through snRNA-RNA and RNA-protein interactions. The U1/5′SS interaction is thought to be additionally stabilized by interactions with the CBC and BBP/mud2; however, the quantitative contribution of each participant is not well understood. Here we use Colocalization Single Molecule Spectroscopy (CoSMoS) to monitor the interaction between fluorescently labeled U1 and pre-mRNA. By selectively disrupting individual CC interactions and monitoring the stability of the U1/5′SS complex we have been able to quantify the contributions from individual CC components. Our results provide the first description of how cross-intron interactions formed between the U1 snRNP and other CC components impact the kinetics of the U1/5′SS interaction. Furthermore, our single molecule results provide kinetic evidence for the synergy between the CBC, BBP/Mud2, and the U1 snRNP: long-lived U1/5′SS association requires the presence of either CBC or BBP/Mud2 even though each individually only makes minor contributions. This is in agreement with a “buffering” model obtained using yeast genetics by Schwer et al. for CC interactions. Finally, we have used this system to dissect the roles of snRNP proteins and the snRNA in U1/5′SS association. Our data is consistent with a multi-step pathway for U1 recruitment to the pre-mRNA that may rely on CC components to efficiently trigger stable U1 binding.