Mosteukaryotic genes contain introns which must be removed from pre-mRNA bythe spliceosome prior to translation. The spliceosome is a mega-Dalton macromolecular machinecomposed of both RNA and protein components. ATP is not utilized for chemical bond formation duringeither lariat synthesis or exon-exon ligation by the spliceosome. Nevertheless, ATP hydrolysis is requiredfor structural rearrangements that are essential for product formation. These structural rearrangementsresult in defined, stable complexes that have been isolated and studied in vitro. It is not known howtransitions between complexes occur, but one hypothesis is that essential DExD/H-box proteins (Prp2,16,22, and 43) initiate structural reorganization of the spliceosome by using the energy of ATP hydrolysis todisrupt protein/RNA or RNA/RNA interactions. Given the ~3 MDa size of the spliceosome, traditional biochemical analysis of how these enzymesfacilitate splicing is difficult. In this research proposal, single molecule fluorescence (SMF) will be used as anew method to study interactions between DExD/H-box proteins and the spliceosome. Spliceosomes will beassembled on a derivitized glass surface from Prp2-deficient yeast cell extract. Dueto the absence of Prp2,the spliceosomes will assemble on a surface-immobilized, fluorescently-tagged pre-mRNA substrate but willbecome stalled prior to lariat formation. The Prp enzymes will be expressed and purified from E. coli using published procedures andfluorescently-labeled using either a genetically-encoded GFPvariant or keto-biotin. Interactions betweenthe enzymes and the immobilized spliceosomes will be visualized using multi-wavelength SMFmicroscopy.These experiments will provide new insight into spliceosome catalysis not attainable with ensemble methodsby unambiguously testing the proposed transient associationof each DExD/H-box protein with thespliceosome and the role of ATP hydrolysis and conserved protein domains in spliceosome binding.
Splicing of pre-mRNA transcripts is an essential step in gene expression, and errors in pre-mRNA splicing have been correlated with a number of cancers including infant brain tumors, breast cancers,and retinoblastomas. Understanding how the spliceosome catalyzes splicing is critical for understandinggene expression in both healthy and diseased eukaryotic cells. Elucidation of DExD/H-boxproteininteractions with the spliceosome using single molecule fluorescence will provide new insight into thechemical steps of splicing not possible with ensemble measurements.
| Anderson, Eric G; Hoskins, Aaron A (2014) Single molecule approaches for studying spliceosome assembly and catalysis. Methods Mol Biol 1126:217-41 |
| Crawford, Daniel J; Hoskins, Aaron A; Friedman, Larry J et al. (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site. Proc Natl Acad Sci U S A 110:6783-8 |
| Hoskins, Aaron A; Friedman, Larry J; Gallagher, Sarah S et al. (2011) Ordered and dynamic assembly of single spliceosomes. Science 331:1289-95 |
| Hoskins, Aaron A; Gelles, Jeff; Moore, Melissa J (2011) New insights into the spliceosome by single molecule fluorescence microscopy. Curr Opin Chem Biol 15:864-70 |
| Crawford, Daniel J; Hoskins, Aaron A; Friedman, Larry J et al. (2008) Visualizing the splicing of single pre-mRNA molecules in whole cell extract. RNA 14:170-9 |
