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

Abstract

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-₄₁AACAAU₄₆ region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

Keywords: Prp16; Prp8; U6 snRNA; spliceosomal catalytic center; transition-state conformation.

FIGURE 1.

Structural representation of spliceosomal components involved in the first-to-second steps transition. (A) Structure of S. cerevisiae spliceosome immediately after branching (5LJ5) (Galej et al. 2016). Spliceosome factors involved in the transition between the first and second steps are shown in color: Prp16 (pink), Isy1 (blue), Ecm2 (light green), U6–U2 helix Ia and the catalytic triplex (red/green), U2 stem IIa/IIc (green), U6-ISL and U6-₄₁AACAAU₄₆ (red), Cwc2 (orange), and Prp8 N-terminal domain (purple). Location of cwc2, U6, and prp8 alleles suppressing prp16-302 growth defects described in this paper are marked by spheres. (B) Schematic representation of U6 (red), U2 (green), and pre-mRNA (gray) at the catalytic center of the spliceosome. Base-pair interactions altered by Prp16 action are depicted.

FIGURE 2.

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.

FIGURE 3.

U6 alleles upstream of ACAGA motif support transition from the first-to-second steps of splicing. (A) Prp16 promotes transition from the first to the second step, whereas prp16-302 allele inhibits this transition. (B) Schematic representation of U6 (red), U2 (green), and pre-mRNA (gray) at the catalytic center of the spliceosome. Mutations of U6 in the ₄₁AACAAU₄₆ region are marked. In prp16Δ U6Δ strain (yCQ166), prp16-302 (C) and prp16-R686I (D) alleles inhibit transition to the second step, exhibiting cs phenotypes suppressed by mutations within the U6 region ₄₁AACAAU₄₆, as shown by spotting on 5FOA plates. (E) Growth curves (OD₆₀₀) of yeast strains harboring prp16-302 allele in combination with U6-A42g and A44c alleles exhibit improved growth, compared to U6-WT at 16°C.

FIGURE 4.

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.

FIGURE 5.

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.

FIGURE 6.

Interactions between various classes of U6 and prp8 N-terminal alleles. (A) The model of rearrangements of the catalytic center between the two catalytic steps. Four classes of alleles are marked by colored boxes: those promoting exit from the first-step catalytic conformation and entry into the transition-state conformation (light red); those promoting entry into the transition-state conformation through destabilization of both catalytic steps (red); those inhibiting exit from the first-step catalytic conformation (light blue), and those promoting both catalytic conformations (blue). (B) Genetic interactions between prp8-T589P, Y590F, K603I, K611T, S613T and U6-A59c, C61a, A79g, G63c, A42g, C43u alleles in prp8Δ U6Δ strain (yCQ05) tested by growth in the presence of 5FOA. prp8-T589P, K603I, K611T, S613T alleles become synthetically lethal in combination with U6-A59c. U6-C61a and A79g become synthetically lethal with prp8-K611T allele or cs and/or ts in combination with prp8-T589P, Y590F, K603I, S613T alleles. prp8-T589P, Y590F, K603I, K611T alleles do not affect growth in combination with U6-G63c, A42g, C43u, compared to wt U6.

Jadwiga Meissner

Katarzyna Eysmont