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

prp8 alleles located in the N-terminal domain suppress prp16 defects. (A) Prp8 N-terminal domain (purple), U6 (red), Cwc2 (orange), and pre-mRNA (intron-black, 5′ exon-yellow) as seen in complex C (5LJ5) (Galej et al. 2016). Locations of prp8, cwc2, and U6 alleles are marked by spheres. The inset: a region of close interactions between Prp8, Cwc2, and U6. In prp16Δ prp8Δ strain (yCQ06), the cs phenotype of prp16-302 (B) and prp16-R686I (C) alleles is suppressed by mutations in the Prp8 N-terminal domain identified in the genetic screen. (D) Growth of combinations of prp8 alleles Y590F + K603I, K603I + K611I, and T589P + K611T is not affected compared to single mutations. Suppression effects in B–D were monitored by spotting of the analyzed strains on 5FOA plates. (E) Growth curves (OD₆₀₀) of yeast strains harboring prp16-302 allele in combination with prp8-Y590F, T589P, K611T/I, and K603I alleles show improved growth compared to Prp8-WT at 18°C. Growth curves of combinations of prp8 alleles Y590F + K603I (F), K603I + K611I (G), and T589P + K611T (H) demonstrate no additive growth improvement of strains carrying prp16-302 allele at 18°C compared to single alleles.