Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing

Abstract

After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.

FIGURE 1.

Genetic interactions between Prp8, Prp22, and the U5 snRNA. (A) Fivefold serial dilutions of prp8-R1753E cells bearing plasmids (URA3 CEN) for expression of wild-type Prp8, wild-type U5 snRNA, and the mutants U5-S1, U5-S2, U5-GG, and U5-CT were spotted to minimal agar medium lacking uracil. The plates were photographed after 3 d of incubation at 30°C, 34°C, and 37°C. (B) prp22-T637A, prp22-S635A, and prp22-H606A were transformed with plasmids expressing wild-type Prp22 and U5 snRNAs as indicated at the left. Serial dilutions (10-fold) were spotted to agar medium and photographed after 3 d of incubation at 30°C and 35°C, and 4 d at 25°C. (C) Alignment of the wild-type U5 loop 1 sequence with those of the suppressors U5-S1 and U5-S2. Mutated nucleotides in the suppressor U5′s are in bold and underlined. The loop 1 sequences in U5-GG and U5-CT are U4G, U5G and U7C, A8U, respectively. (D) Model for interactions between the U5 loop and exon sequences prior to step 2 (left) and after exon joining (right). The dashed lines indicate putative base-pairing interactions between the U5 loop 1 and exon sequences (Newman 1997). Exons 1 and 2 are drawn as gray and black rectangles, respectively; the intron is indicated by a line. Loop 1 nucleotides, which are numbered 1–9 according to (Newman and Norman 1991), correspond to nucleotides 93–101 in the U5 snRNA (Frank et al. 1994).

FIGURE 2.

(A) Splicing of three exon-mutant substrates in vitro using extracts from wild-type PRP8 or mutant prp8 cells. ACT1 substrate with the exon mutations shown at the top of each panel was spliced in vitro in wild-type PRP8 extract (wt), in prp8-R1753K extract (R→K), or in prp8-R1753E extract (R→E), as indicated above each panel. Aliquots were withdrawn at the times indicated along the top of each gel and analyzed by denaturing PAGE. Autoradiographs of the gels are shown. The positions of the pre-mRNA, splicing intermediates, and mRNA are depicted at the left. (B) Relative molar amounts of RNA species derived from the wild-type (lanes 1–3) and prp8-R1753E (lanes 7–9) time courses are graphed for the AAAA|AAA pre-mRNA. The pre-mRNA is represented by squares, lariat intermediate by circles, and mRNA by triangles; the dashed lines represent wild-type extracts and the solid lines prp8-R1753E extracts. (C) Graphical representation of the relative amounts of the intermediates/mRNA for the 9 min time points (wt extract: lanes 2,11,20; for R→K: lanes 5,14,23; for R→E: lanes 8,17,26). The number beneath each bar indicates the pre-mRNA: 1: AAAA|AAA; 2: AAAC|CAA; and 3: CTTC|CAA.

FIGURE 3.

Gain-of-function mutations in Prp18. (A) Serial 10-fold dilutions of prp8-R1753E cells bearing plasmids that carry the PRP8 gene or various PRP18 alleles as indicated at the left were plated to agar medium lacking uracil. Cells containing the empty plasmid (vector) were analyzed in parallel. The plates were photographed after 3 d of incubation at 30°C and 37°C. (B) A photograph of slu7-EIE cells containing the indicated expression plasmids after 4 and 2 d of incubation at 25°C and 37°C, respectively, is shown. (C) Stereo representation of Prp18-(80–251) shown as a ribbon diagram (Jiang et al. 2000). Ser162 and Val191are shown as sticks.