SNRNP-SUBSTRATE INTERACTIONS IN S POMBE SPLICING

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DESCRIPTION: (Adapted for applicant's abstract): The site of nuclear pre-messenger RNA splicing is a complex structure the size of a ribosomal subunit known as the spliceosome. Many of its components remain to be identified, and only a small subset have been characterized at the molecular level. While the general mechanism of splicing is conserved through evolution, more detailed studies have revealed differences both in the structures of the snRNAs which lie at the heart of the machinery and in the signals they recognize in the substrate, among others. The applicant has embarked on an analysis of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe because its vast phylogenetic separation from organisms in which splicing has been more extensively studied makes it ideal for distinguishing those facets universal to all eukaryotes from other, group-specific, specializations of the ancestral mechanism. An additional advantage of this organism is its amenability to genetic manipulation both by classical and modern methods. The applicant will focus most intensively on exploring all aspects of U1 snRNP function. First, in vitro mutagenesis of the U1 snRNA gene and model splicing substrates followed by transformation and phenotypic analysis will be used to characterize their interactions; in addition to providing phylogenetic perspective on differences in 5' junction recognition observed by previous investigators, these studies will test a model the applicants have developed for hydrogen bonding of U1 to the 3' junction. An open-ended mutagenesis strategy will allow the investigator to identify residues of the U1 snRNA proteins important for spliceosome assembly and perhaps other, as yet unsuspected, functions. Suppression of splicing defects that cannot be fully rescued by compensatory mutations in an snRNA will be used to identify non-snRNP factors that interact with splicing signals. Secondary goals of the program will be to examine the evolutionary origin and functional significance of the intron in the U6 snRNA gene, and the significance of a potential interaction of U5 snRNA with the 5' splice site. Interactions identified on the basis of in vivo experiments will be examined in detail using a homologous in vitro splicing extract, which will allow us to observe intermediate steps in spliceosome assembly in order to determine the biochemical basis of mutant phenotypes.