Mutational Analysis of pre - mRNA Splicing in Saccharomyces cerarisiae Using a Sensitive New Reporter Gene , CUPl Cammie F . Lesser

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An introncontaining fragment from the Saccharomyces cerevisiae ACTl gene has been fused to C U P l , the yeast metallothionein homolog. CUPl is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACTl-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U 1 snRNA. The use of CUPl as a reporter gene may be generally applicable for monitoring cellular processes in yeast. T HE goal of this work was to develop an improved genetic system by which to monitor pre-mRNA splicing in vivo. Intron-containing fragments of several Saccharomyces cerarisiae genes have previously been fused to reporter exons, including @-galactosidase ( L a d ) from Escherichia coli (LARSON et al. 1983; TEEM and ROSBASH 1983), HIS4 from S. cerevesiae (PARKER and GUTHRIE 1985) and thymidine kinase from the herpes simplex virus (FOUSER and FRIESEN 1986). These gene fusions have been used (1) to monitor the effects of intron mutations on the production of mature mRNA by measuring the activity of the fusion protein and (2) to genetically identify splicing components involved in intron recognition. T o date, there have been only two reports of the successful use of the reporter genes for the genetic identification of trans-acting splicing components [ACTI-HIS# and ACTI-Lac2 gene fusions (COIJTO et al. 1987) and CYH2-Lac2 gene fusions (NEWMAN and NORMAN 199 l)]. The level of sensitivity and the range of detection of gene fusion reporter products are two very important criteria for the design of useful genetic schemes. HIS4 gene fusions have limited genetic utility for both of these reasons. First, a high threshold concentration of HIS4 fusion protein needs to be produced in the cell before any growth on media containing histidinol (the biosynthetic precursor of histidine) can be detected (PARKER and GUTHRIE 1985; VIJAYRAGHAVAN et a l . 1986). Second, once the threshold level is obGenetics 133: 851-863 (April, 1993) tained, only a small range of increasing amounts of HIS4 fusion protein can be distinguished before the protein is no longer limiting for growth (VIJAYRAGHAVAN et al. 1986). In contrast, while LacZ fusions are very sensitive and provide relatively linear indicators of splicing efficiency, these reporters fail a third criterion: they cannot be used for the direct selection of suppressors; rather the &galactosidase activity of each mutant candidate must be screened. In order to avoid many of these limitations, we have designed a new gene fusion using CUPI as the reporter. CUPl is normally a nonessential gene (HAMER, THIELE and LEMONTT 1985), but it allows cells to grow in the presence of otherwise lethal concentrations of copper by chelating the metal in a dosagedependent manner. CUPl has been demonstrated to provide a sensitive assay for studying gene amplification (WELCH et al. 1983; FOGEL et al. 1983). The threshold level of detectable CUPl protein in a cell is very low and there is a large range (0.1-6.0 mM CuSO4) over which different copper concentrations can be distinguished by monitoring growth on coppercontaining media (FOGEL et al. 1983; HAMER, THIELE and LEMONTT 1985). In order to facilitate our studies of mRNA splicing, we constructed a gene fusion by combining the 5' exon and intron-containing region of the yeast A C T l gene with the complete coding sequence of 'CUPI. In this way we are able to study the efficiency of pre-mRNA splicing by monitoring growth on copper-containing media. 852 C. F. Lesser and C. Guthrie