FIGURE 2.

Screen for synthetic enhancers of L30 repression of splicing. (A) Reporter plasmids based on the fusion between RPL30 exon 1 and intron with the CUP1 ORF. LCUPIF transcripts (left) produce Cup1 protein only when unspliced, while LCUP RNAs (right) need to be spliced to encode the protein. (B) Phenotype, identified as growth in medium-containing copper, of cells with constitutive excess of L30 (yJV25) and transformed with either pLCUPIF (upper panel) or pLCUP 5A (bottom panel). Under repression conditions (“+” rows), pLCUPIF confers copper resistance while pLCUP-5A does not. When L30 repression is abolished by the C9U mutation (“−” rows), pLCUP confers tolerance while pLCUPIF does not. Serial one-fifth dilutions were spotted in each case. (C) Screen strategy to select mutations that restore inhibition of splicing by L30 on a C9U transcript. Corresponding Northern analyses are shown in panels D and E. Strain yJV25 with the plasmid pLCUPIF-C9U (Cu-sensitive) was UV-irradiated and SLR mutants were selected on plates containing 0.3 mM copper. Colonies showing LCUPIF C9U pre-mRNA accumulation (panel D) were cured of the plasmid and transformed with pLCUP 5A C9U, rendering them Cu sensitive again because of increased repression, unless the slr mutation is complemented or suppressed. Thus, cells were transformed with a YCp50-based wt genomic library and the transformants selected on 0.7 mM copper, and pCBP80 was identified (panel E). (D) Northern analysis of RNA from SLR mutant cells transformed with pLCUPIF C9U (lanes 3–8). As controls, RNA extracted from yJV25 cells harboring pLCUPIF wt (lane 1) or C9U (lane 2) were loaded in the same gel. Precursor (p) and mature (m) LCUPIF transcripts are indicated. (E) Northern analysis of RNA extracted from SLR4, SLR5, and SLR7 (lanes 3–8) and yJV25 cells (lanes 1,2), transformed with pLCUP 5A C9U alone (odd lanes), or plus pCBP80 (even lanes). Precursor (p) and mature (m) LCUP transcripts are indicated. In D and E U3 was used as loading control.