Function of Snrnp Particles in Premrna Splicing

Rymond, Brian

Abstract

This is a revised renewal application to study interactions between U1 snRNP and other components of the splicing machinery. The major focus is on a U1-associated protein, Prp39, identified by the investigator in an earlier screen for novel ts splicing mutants. Based on the observations that this protein 1) is required for formation of commitment complexes, 2) yet is only weakly associated with U1 snRNPs, and 3) associates with unspliced precursor but not intermediates, the investigator will test a central hypothesis that U1 snRNP recruitment to and subsequent release from the spliceosome is mediated by the association and dissociation of weakly bound proteins. A major goal is to determine the precise times in the cycle at which Prp39 and other known U1-snRNP proteins (Snp1 (70k), mud1, mud2) associate and dissociate from U1 and from the spliceosome; this approach relies on the availability of antibodies and of a collection of prp mutations blocked at specific steps in the pathway. Crosslinking experiments will address the hypothesis that Prp39 stabilizes U1 binding to the 5' splice site by direct interaction with the pre-mRNA and determine at what point the U1/5' SS interaction is replaced by the U6/5'SS interaction. Interactions between Prp39 and other U1 snRNP proteins will be sought by the 2-hybrid approach. Sequences in U1 necessary for Prp39 interaction will be identified by mutagenesis. A mammalian homologue will be sought by immunological and genetic means; this will allow a test of the prediction that it is a non-snRNP splicing factor in higher organisms. The role of Prp39 in maturation of the 3' end of U1 snRNA will be analyzed. Finally, a genetic screen is described to allow identification of genes that enhance splice site selection.