Direct Observation of pre-mRNA Arrangements During Spliceosome Assembly Using Single Molecule FRET

Abstract

In eukaryotes, transcription of genomic DNA produces precursors to messenger RNAs (pre-mRNAs) which contain both expressed regions (exons) and intervening non-coding regions (introns). Pre-mRNA splicing, the process of exon ligation with concurrent intron excision via two transesterification reactions, is carried out by the spliceosome: a highly dynamic macromolecular complex consisting of five core small nuclear ribonucleoprotein particles (snRNPs) and many transiently associated proteins. We used single molecule fluorescence resonance energy transfer (FRET) and single molecule fluorescence colocalization (CoSMoS) methods to detect pre-mRNA structural rearrangements during the splicing reaction and to investigate their timing with respect to the steps of spliceosome assembly in budding yeast (Saccharomyces cerevisiae). We first prepared a model pre-mRNA containing two site-specifically incorporated fluorophores. The FRET acceptor Cy5 was placed within the 5′ exon near the 5′ splice site, whereas the FRET donor Cy3 was positioned just upstream of the intron branch point nucleotide that provides the nucleophile for the first transesterification reaction. Then, by monitoring changes in FRET efficiency on single pre-mRNAs during splicing, we observed conformational changes within the pre-mRNA. Further, by stalling the spliceosome at defined points during the reaction, discrete FRET efficiencies were correlated with association of particular snRNPs. Collectively, these experiments are providing insight into the stages of spliceosome assembly and activation at which the branch site and 5′ splice site come into spatial proximity, a necessary precursor to catalysis.