Splice site selection in Saccharomyces cerevisiae pre-mRNA splicing

Sammanfattning: Pre-mRNA splicing is the process by which intervening sequences (introns) are removed from the coding sequences (exons) resulting in a mature messenger-RNA (mRNA). Splicing takes place in a large ribonucleoprotein complex termed the spliceosome, composed of several protein components and the five small nuclear RNAs (snRNAs) U I, U2, U4, U5 and U6. Genes of higher eukaryotes may contain more than 50 introns, each of which has to be accurately removed to yield a functional mRNA. The exon sequences are distinguished from the introns by short, conserved sequence elements within the intron. These sequences are known as splice sites. The goal of this work has been to investigate the components and mechanisms involved in selection of pre-mRNA splice sites. As a model system to study the process of intron removal, the budding yeast Saccharomyces cerevisiae was used. The well developed genetic and biochemical tools available in S. cerevisiae offer a unique opportunity to directly study the role of individual splicing factors on selected pre-mRNA reporter constructs in vivo. Data obtained in this simple eukaryotic system can be applied to higher eukaryotes, as the mechanism and components of pre-mRNA splicing are phylogenetically conserved. In the first paper the selection of the first two nucleotides of an intron was investigated. This GU dinucleotide is conserved in almost all naturally occurring introns, and previous work in the laboratory had established that a component of the splicing machinery recognized the GU before the first step of splicing. We identified a region of the U6 snRNA as being involved in selection of the first and third intron nucleotides for the first step of splicing. In addition, we could demonstrate that mutations in said region of U6 snRNA displayed different splicing phenotypes depending on what reporter construct was studied. In the second paper an in vivo regulated U6 snRNA transcription unit was constructed. U6 snRNA is transcribed by RNA polymerase III in yeast, and is not amenable to regulation by conditional polII promoters. Based on expression of the E.coli lac repressor protein (lacI), we successfully repressed transcription from a U6 snRNA gene harboring the lacI binding site in the promoter region. The conditional transcription system was used to investigate a dominant negative U6 snRNA mutation. It was demonstrated that a single point mutation in U6 snRNA position A51 could confer a dominant lethal phenotype, and that in these cells splicing was blocked after the first step. In the third paper we investigated the role of an RNA structure known as U2/U6 helix Ib in 5' splice site selection. The nucleotide of U6 snRNA (G52) that selects the first intron nucleotide has been postulated to interact with a U2 snRNA residue (A25). Mutational analysis of U2-A25 and surrounding residues revealed that mutations disrupting U2/U6 helix Ib negatively affected 5' splice site cleavage. Restoring the helical structure restored splicing, however, U2 residue A25 was not involved in 5' splice site selection. These data suggest that the first intron nucleotide may be selected through multiple sequential or redundant interactions with the splicing machinery. In the fourth paper the mechanism and components of 3' splice site selection was investigated. A direct, non Watson-Crick interaction between intron terminal nucleotides has been suggested to play a role in selection of the last intron nucleotide. Using a reporter construct with two closely spaced, competing 3' splice sites we could demonstrate that the spliceosome was far more tolerant to variations in the first and last intron positions than was previously reported. These data indicated that any interaction between intron terminal nucleotides was likely to be indirect. We could demonstrate that the branch point - 3' splice site distance was a crucial factor in 3' splice site selection. ISBN 91-628-2872-X