Reconstitution and Single Molecule Characterization of Yeast Spliceosomal E Complex

Abstract

The formation of yeast commitment complex (CC2 or “E complex”) is one of the earliest stages in spliceosome assembly on pre-mRNA, and is the only assembly step that forms independent of the activity of an ATPase. Previous studies have shown that E complex components, the nuclear cap binding complex (CBC) and the branchpoint binding Msl5•Mud2 heterodimer, are required to stabilize U1 small nuclear ribonucleoprotein (snRNP) complex to the 5′SS of pre-mRNA. However the kinetics and nature of the interactions between these components have not been established, in part because E complex has never been reconstituted separate from other spliceosomal proteins. Here we present work towards the characterization of E complex interactions by Colocalization Single Molecule Spectroscopy (CoSMoS) experiments with reconstituted fluorescently-labeled CBC, Msl5•Mud2, and U1 snRNP. Intact and functional U1 snRNP was isolated from all other spliceosomal proteins by a novel purification method and fluorescently labeled with a tri-functional SNAP-ligand derivative for single molecule study. The stoichiometry of the seventeen U1 snRNP proteins and the efficiency of the fluorescent labeling reaction is being determined by AQUA mass spectrometry. A combination of splicing assays, primer extension, and CoSMoS experiments with fluorescently labeled pre-mRNA constructs are used to show the functionality of purified U1 snRNP. Our results show that the multi-protein components of E complex can be reconstituted and fluorescently tagged for CoSMoS and other single molecule experiments which elucidate the kinetics of early spliceosome assembly.