Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -actingfactors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of thesefactors into a large complex, the spliceosome, is essential for the subsequent two stepsplicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate(intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligatedand lariat intron released. A combination of genetic and biochemical techniques have beenused here to study pre-mRNA splicing in yeast.
Yeast introns have three highly conserved elements. We made point mutationswithin these elements and found that most of them affect splicing efficiency in vivo and invitro, usually by inhibiting spliceosome assembly.
To study trans -acting splicing factors we generated and screened a bank of temperature-sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) wereisolated. The four phenotypic classes obtained affect different steps in splicing and accumulateeither: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNAand intron. The latter three classes represent novel phenotypes. The excised intron observedin one mutant: prp26 is stabilized due to protection in a snRNP containing particle.Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate andexon 1. This is especially interesting as it allows analysis of the second splicing reaction.In vitro complementation of inactivated prp18 extracts does not require intact snRNPs.These studies have also shown the mutation to be in a previously unknown splicing protein.A specific requirement for A TP is also observed for the second step of splicing. ThePRP 18 gene has been cloned and its polyadenylated transcript identified.
