Effects of U6, cwc2, and prp8 alleles on splicing of suboptimal introns. (A) Schematic of ACT1–CUP1 reporter indicating the used 5′SS, BS, and 3′SS mutants. (B–D) Primer extension and copper growth assays for intron mutant reporters in strains carrying U6 (yMK20), cwc2 (yMK79), or prp8 (yJU75) alleles. (NC) Not calculated. (B) cwc2-W37A and S38P alleles exacerbate splicing defects of BS-c reporters (NC: bands corresponding to lariat-intermediate and spliced mRNA products are not detectable, preventing calculation of splicing efficiency) and improve the second step of splicing of A3c reporters. (C) U6-A42g and C43u alleles exacerbate the first step of splicing of the BS-c reporter while improving the second step of splicing of the A3c reporter (U6-A44c and A45g alleles display a similar, though very modest effect). (D) prp8-T589P, K603I, K611I, S613T alleles inhibit splicing of BS-c (for K603I, K611I, and S613T, alleles bands corresponding to lariat-intermediate and spliced mRNA products are not visible, preventing calculation of splicing efficiency) and A3c reporters. prp8-V1870N and W1575R second-step alleles improve splicing of gAG/reporters and prp8-K603I inhibits it. prp8-K603I allele opposes prp8 second-step W1575R allele, diminishing its improvement of gAG/intron splicing. Only the first step of gAG/intron splicing is inhibited by prp8-K603I + V1870N allele compared to prp8-V1870N allele.
