Mutation of the ZnF domains of Luc7 can have specific impacts on 5′ss usage. (A) Schematic representation of the ACT1-CUP1 reporter pre-mRNA and splicing assay. Proper processing of the splicing reporter confers resistance to Cu2+ in the growth media. (B) ACT1-CUP1 assays using a reporter with substitutions in the 5′ss sequence U2A (GaAUGU) and the indicated changes in the exon. ZnF1 mutants have no obvious effect on splicing. Dots represent the maximum [Cu2+] tolerated in two independent replicates. (C) Same as in B but using a reporter with the 5′ss G5A (GUAUaU). (D) ACT1-CUP1 assays using ZnF2_L/L2 to assay intronic positions of the 5′ss. Mutation of Luc7 ZnF2 improves splicing of A3U, G5A, G5C, and G5U but does not affect other reporters. (E) ACT1-CUP1 assays using the NΔ31 truncation mutant of Luc7.
