Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing

cwc2 alleles identified in a genetic screen alter the first-step conformation, supporting transition to the second step. (A) Schematic of the catalytic phase of splicing. Conformations of the catalytic center are shown in brackets, and factors affecting transitions between these conformations are listed above (promoting) or below the arrows (inhibiting the transition). (B) Schematic of the yeast Cwc2 protein domain structure. Positions of alleles identified in the genetic screen are marked. (C) cryo-EM structure of Cwc2 (orange) and part of U6 snRNA (red) within the spliceosome complex C (5LJ5) (Galej et al. 2016). Positions of cwc2 alleles are marked as spheres. The inset shows two Cwc2 amino acids, W37 and S38, adjacent to U6 C43-G39. In prp16Δ strain (yMK36), the cs growth phenotype of (D) prp16-302 and (E) prp16-R686I alleles is suppressed by the identified cwc2 alleles, as shown by spotting on 5-fluoroorotic acid (5FOA) plates. (F) OD₆₀₀ measurements of yeast strains harboring prp16-302 allele and a second copy of cwc2-W37A and S38P alleles show an improved growth as compared to Cwc2-WT at 16°C. (G) In prp2Δ strain (yAAH1915), cwc2 alleles W37A and Q54L exacerbate the prp2-Q548N cs phenotype, as shown by spotting on 5FOA plates.