Development of a novel screening method for the isolation of precursor RNA processing mutants of Saccharomyces cerevisiae
The availability of conditional-lethal yeast (Saccharomyces cerevisiae) mutants that are defective in the process of pre-mRNA splicing (precursor RNA processing; prp) has greatly facilitated the characterisation of components of the splicing machinery. When this project was initiated nine pip complementation groups had been defined (prp2-prpll), all of which accumulate pre-mRNA at the expense of mRNA when incubated at the non-permissive temperature. Pre-mRNA splicing is a complex and dynamic process relying on many gene products for its completion and therefore many more complementation groups remained to be identified. Determination of a prp phenotype has relied on the direct measurement of protein to RNA ratios and on the Northern blot analysis of conditional-lethal mutants incubated at the restrictive temperature. Such analyses are both time-consuming and labour-intensive. In this thesis I present the development of a novel screening procedure which positively identifies prp mutants. I have fused a yeast intron-containing gene to the lacZ gene of E.coli such that only the pre-mRNA generated from this fusion can encode an active β-galactosidase fusion-protein. This gene-fusion has been introduced into a prp2 strain and the encoded pre-mRNA has been shown to accumulate on incubation at the non-permissive temperature. This pre-mRNA accumulation results in an increase in β-galactosidase activity. Exploiting this observation a simple plate assay has been developed and used to screen a pool of temperature-sensitive mutants for defects in pre-mRNA splicing. A number of prp mutants have been identified and I present the results of their initial characterisation.