Single Molecule Fret Characterization of Pre-Mrna Splicing: Substrate Dynamics During Recognition and Catalysis

The spliceosome is a complex ribonucleoprotein (RNP) that catalyzes the removal of intervening sequences (introns) from coding regions (exons) in eukaryotic pre-mRNAs. The ordered assembly of the spliceosome makes it a compositionally dynamic system where protein and RNA components are shuttled in and out in a highly regulated manner. Furthermore, catalytic activation of the spliceosome requires a dynamic set of ATP dependent RNA-RNA and RNA-protein interactions. Extensive genetic and in vitro biochemical work has been done to define the cis- and trans- acting factors that are required for both steps splicing. On the other hand, the pre-mRNA substrate which acts as both the scaffold for assembly and is a key component of the chemical steps after activation by a yet to be identified spliceosomal catalyst has been wholly ignored thus far. The difficulty in interrogating this system without synchrony and inactivation, thus precluding real time measurements, has proven difficult to overcome. To dissect the kinetic and conformational requirements for pre-mRNA positioning during spliceosome assembly we developed an in vitro single molecule FRET (smFRET) splicing assay in which the position of conserved intronic sequences have been tracked in real-time throughout splicing. The ability to stall the splicing process with mutations in either the pre-mRNA or splicing components we can dissect the role of spliceosome components in regulating pre-mRNA conformations. Additionally, we have utilized an affinity purification technique to determine the relative positions of conserved sequences in isolated splicing intermediates. We have observed reversible conformations changes whose kinetics are affected by ATP and the identity of splice sites.The kinetics of these conformations has implications for proofreading and catalysis that are essential for the maintenance of proper gene expression.