Preparation of Fluorescent Pre-mRNA Substrates for an smFRET Study of Pre-mRNA Splicing in Yeast

The spliceosome is a complex small nuclear (sn)RNA–protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. For each splicing event, the spliceosome is assembled de novo on a pre-mRNA substrate and a complex series of assembly steps leads to the active conformation. To comprehensively monitor pre-mRNA conformational dynamics during spliceosome assembly, we developed a strategy for single-molecule FRET (smFRET) that utilizes 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. In this chapter, we describe the identification of Ubc4 pre-mRNA that is efficiently spliced in vitro and the methods we have developed for the chemical synthesis of fluorescent Ubc4 pre-mRNA for smFRET.