Using the simple eukaryote Saccharomyces cerevisiae, we have recently succeeded in identifying a set of five small nuclear RNAs (snRNAs) that mediate pre-messenger RNA splicing. We have definitively established a 1:1 correspondence between these spliceosomal RNAs of yeast, and U1, U4, U5, and U6 snRNAs of mammalian cells, despite surprising differences in size and structural organization. These data have provided unexpected insights into the evolutionary constraints operating on the spliceosomal RNAs, revealing a functional hierarchy which places U6 at the center of this RNA-based machine. Toward the long-term goal of determining the three-dimensional structure of the spliceosome, we will now proceed to augment this information by the establishment of a larger phylogenetic data base comprised of snRNA sequences from selected fungal representatives. In conjunction with chemical and enzymatic conformational probing, phylogenetically derived structural models will also be tested using genetics. Deletion analyses will be used to delineate absolutely required structure: function domains; since our initial forays have demonstrated that even phylogenetically conserved elements may perform functions dispensable for viability, we anticipate generating a collection of "mini-snRNAs" smaller than any found in nature. In a complementary approach, we will assess the contributions of species-specific and evolutionarily invariant domains by performing inter-species "swaps", using engineered chimeras to complement deletion strains in yeast. Beyond their obvious impact on a mechanistic understanding of splicing, by focusing on the RNA-based functions of the spliceosomal machinery these approaches will also provide an experimental avenue to explore the provocative hypothesis that the trans-acting snRNAs once resided within the introns of self-splicing RNAs.
