Genetic and functional interaction of evolutionarily conserved regions of the Prp18 protein and the U5 snRNA

Abstract

Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.

FIG. 1.

Locations of mutations in yeast Prp18. (A) Amino acid sequence of S. cerevisiae Prp18. The positions of α-helices (black boxes labeled 1 through 5) and loops between them (lines connecting the boxes) are shown above the sequence, as determined from the X-ray crystal structure of a large fragment of Prp18 (32). The most conserved region of Prp18 from S187 to I211 is boxed in the sequence. The positions of most of the mutations in Prp18 proteins used in this study are shown. (B) List of Prp18 mutants used; all were described by Bačíková and Horowitz (2).

FIG. 2.

snr7 suppressors of prp18ΔCR. (A) Bases 85 through 109 of S. cerevisiae U5 snRNA are shown in their canonical stem-loop structure (21). The numbering of G93 through C101 as bases 1 to 9, which we use throughout the text, is indicated (37). The positions and changes of the U5 snRNAs transcribed from the suppressors are shown, together with the frequency with which each position was mutated to the nucleotide shown. (B) The sequences of the suppressors are shown, with only positions 2, 4, and 8 displayed, since all other positions are wild type, together with the number of occurrences of each suppressor.

FIG. 3.

Growth of prp18ΔCR and wild-type yeast bearing U5-loop 1 suppressors. Yeast with a HIS3-disrupted PRP18 were transformed with a plasmid bearing prp18ΔCR or PRP18 (indicated at the left) plus a plasmid with a mutant of SNR7 (U5 snRNA) (shown under the U5 heading immediately left of the photographs). Yeast were spotted in fivefold dilutions and grown for 5 days at 23°C, 4 days at 26°C, 3 days at 30 or 34°C, or 3 to 5 days at 37°C, as indicated at the top. The yeast shown here had a wild-type, chromosomal copy of SNR7.

FIG. 4.

Allele specificity of U5-loop 1 suppressors. Assays of seven prp18 alleles with two representative suppressors are shown. Yeast with a HIS3-disrupted PRP18 were transformed with a plasmid bearing the prp18 mutant (or wild-type) allele, indicated at the left under the prp18 heading and with a second plasmid bearing the SNR7 (U5 snRNA) allele indicated at the top of the photographs. The prp18 alleles used are shown in Fig. 1. Yeast were streaked and grown at the temperature indicated at the top for 4 days at 37°C and as stated in the legend to Fig. 3 at other temperatures.

FIG. 5.

Growth of yeast with a single mutant allele of SNR7 (U5 snRNA gene) with a prp18ΔCR or wild-type PRP18 allele. Haploid snr7::KAN prp18::HIS3 yeast carrying a mutant or wild-type SNR7 plasmid were transformed with a plasmid bearing prp18ΔCR or PRP18 as indicated at the left. The yeast were spotted in fivefold dilutions and grown as described in the legend to Fig. 3. Yeast whose U5 is indicated as wt + A8C had both a chromosomal wild-type and plasmid-borne snr7-A8C for comparison with Fig. 3.

FIG. 6.

Point mutations in loop 1 of U5 snRNA. The six mutant versions of U5 snRNA shown were expressed in prp18ΔCR yeast. The snr7-U5A and snr7-U6A alleles, whose mutations are boxed in the figure, were synthetic lethal with prp18ΔCR despite the presence of a wild-type SNR7 allele in the yeast.

FIG. 7.

RNA levels in prp18 mutants. (A) RNAs were detected by Northern blotting of denaturing agarose gels of total RNA extracted from a wild-type strain, four prp18 mutant strains, and a prp18 knockout strain in which Slu7 was overexpressed, as indicated at the top of blots. The prp18 mutants are shown in Fig. 1. Yeast were grown either at 26°C, a permissive temperature for all the strains, or at 26°C with a 2-h shift to 37°C, a restrictive temperature, as indicated at the top of the panel. For ACT1, the pre-mRNA (1,750 bases) and the lariat-exon 2 intermediate (1,600 bases) were detected with an intron-specific probe, and the mRNA (1,450 bases) was detected with a full-length probe (59). The faint band for the 309-base released intron is not shown. CYH2, TDH1, and SEC4 mRNAs were detected with full-length probes, and SCR1 RNA was detected with an oligonucleotide probe. 18S rRNA was detected by staining with ethidium bromide. (B) The ratio of ACT1 intermediate to mRNA was determined from quantitation of Northern blots of RNA extracted from the indicated prp18 mutant strains grown at 26°C or shifted to 37°C. The prp18 mutants are shown in Fig. 1. The units on the y axis are arbitrary and do not represent the actual i/m ratio. The ACT1 lariat-intermediate runs just above the mRNA, and the intermediate would be visible in the section of the blot shown in panel A if there were enough to see.

FIG. 8.

Splicing in prp18ΔCR yeast with snr7 suppressors. (A) ACT1 lariat intermediate (ACT1-int), ACT1 mRNA, and TDH1 mRNA were assayed by Northern blotting of total RNA isolated from PRP18 (wild-type) or prp18ΔCR yeast with a wild-type SNR7 or suppressor snr7-A8C or snr7-U4A allele on a plasmid together with a chromosomal wild-type SNR7 gene. The PRP18 allele, the plasmid-carried form of U5, and the temperature of growth are indicated at the top. Yeast were shifted to 37°C for 2 h. The three RNA species, plus the ACT1 pre-mRNA (ACT1-pre), are indicated in the three panels, which show sequential probings of one blot with ACT1 intron, ACT1 full-length, and TDH1 full-length probes. (B) The ratios of ACT1 lariat intermediate to ACT1 mRNA, determined from quantitation of blots in panel A, are shown. The histogram is aligned with panel A and shares the key at the top of the figure. The units on the y axis are arbitrary and do not represent the actual i/m ratio. Representative data from one experiment are shown here; in the text averages from at least three measurements are used.