Characterization of the Role of PSF During pre-mRNA Splicing

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Patton, James G.NSF MCB-9974542AbstractCharacterization of the Role of PSF During pre-mRNA Splicing. PTB-Associated Splicing Factor (PSF) is a 100kD RNA binding proteinthat was originally identified based on its co-purification withPolypyrimidine Tract Binding Protein (PTB or hnRNP I). Depletion of PSFfrom splicing extracts causes a block to the second step of splicing whichcan be rescued by the addition of recombinant PSF suggesting that PSF is anessential second step splicing factor. To further characterize PSF, aseries of experiments have been performed to analyze its RNA bindingspecificity and identify protein-protein interaction partners. Preliminaryresults suggest that PSF binds to a phylogenetically conserved stemstructure within U5 snRNA (stem 1b). One of the goals of this project isto determine the affinity of PSF for RNAs encompassing one or both strandsof stem 1b followed by direct analysis of PSF-U5 interaction and mapping ofthe exact binding site for PSF on U5. From mutational analyses, it appearsthat the sequence of stem 1b is important not only for the formation of thestem itself, but also as a binding site for a specific protein(s). Theseresults suggest that PSF binds to stem 1b of U5 and that disruption ofPSF-U5 interaction results in a block to the second step of splicing. Todirectly test this hypothesis, a series of quantitative PSF-U5 RNA bindingassays will be performed using fluorescence spectroscopy followed by afunctional in vitro assay in which U5 snRNA will be depleted from splicingextracts and then reconstituted with either wild type or mutated U5 snRNAs.This will allow us to test the effects of specific U5 mutations on bothsplicing and PSF binding. These experiments will be complemented by invivo analyses following the creation of cell lines in which both alleles ofPSF are disrupted and replaced by an inducible PSF transgene. Thus, all U5snRNA mutations that disrupt PSF binding will be tested for their effectson splicing both in vitro and in vivo. Expression of most higher eukaryotic genes requires thatintervening sequences (introns) be efficiently and accurately excised toallow translation of functional proteins. Removal of introns occurs in twosteps within the spliceosome, a large complex containing multiple proteinand protein-RNA complexes. Among the best characterized of the spliceosomecomponents are the small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6,which play central roles both in spliceosome assembly and catalysis. Muchless is known about the protein components of the spliceosome althoughseveral factors have been identified, particularly those required for thefirst step of splicing. In contrast, the identification and functionalcharacterization of proteins required for the second step of splicing haslagged considerably, particularly in higher eukaryotes. The broad goals ofthis project seek to increase knowledge about the second step of splicingin humans and determine whether association of PSF with U5 snRNA isessential for this process.