Conformational dynamics of single pre-mRNA molecules during in vitro splicing

The spliceosome is a complex small nuclear RNA–protein machine that removes introns from pre-mRNAs. A single-molecule fluorescence resonance emission transfer (FRET) assay using the efficiently spliced yeast pre-mRNA, Ubc4, reveals many reversible, time- and ATP-dependent changes in conformation for individual pre-mRNAs and indicates that spliceosome assembly occurs close to thermal equilibrium. The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro , we observed a multitude of generally reversible time- and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3′–splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.