Prp8 forms extensive cross-links to RNA residues in the intron consensus sequences, and in the 5′ and 3′ exons. Intron consensus sequences that define the 5′SS, BP, and 3′SS are indicated. For most yeast introns, a stretch of Pyr, functions with the 3′SS YAG in definition of the 3′SS. Triangles denote the location of Prp8 cross-links observed in either mammalian (▴), or yeast (▵) systems. For all studies except those with an asterisk (*), the location of the cross-linked site is determined through the use of a site-specific photoreactive substitution or by RNase fingerprinting. (*) Approximated sites of cross-linking within an RNase T1 fragment.

Identification of prp8 alleles that suppress mutations in position 2 of the 5′SS. (A) Strategy for isolation of new prp8 alleles. The coding region of PRP8 was divided into four equal fragments (A,B,C,D). Each fragment was amplified by mutagenic PCR and cotransformed with an appropriately gapped plasmid (as shown for B), to generate mutant alleles of prp8 by in vivo gap repair. The resulting strain, which is deleted for its chromosomal copy of PRP8 and CUP1, is diagrammed. Mutant prp8 transformants were screened for the ability to confer increased copper growth on ACT1–CUP1 reporters containing U2A or U2G mutations in the 5′SS, both before and after loss of wild-type PRP8 by 5-FOA selection. (B) Suppression of the copper growth phenotype of mutations in 5′SS position 2 by the newly identified prp8 alleles. Each column shows growth on the concentration of copper, 0.075 mm and 0.05 mm, that is limiting for 5′SS U2A and U2G reporters, respectively. Equal numbers of log phase cells from each strain were spotted onto the same copper-containing plate.

5′SS position 2 suppressor mutations cluster in four regions of PRP8. The location of mutations that confer 5′SS position 2 suppression are indicated in black; green and blue indicate the location of previously characterized 3′SS YAG suppressors and Pyr alleles, respectively (Umen and Guthrie 1996). The corresponding location of the 5′SS GU cross-link in mammalian Prp8 is indicated by the lightning bolt. Solid lines indicate the location of single mutations that confer suppression. Broken lines indicate mutations that confer suppression together but that have not been assayed on their own.

Allele specificity analysis reveals overlapping phenotypes at 5′SS and 3′SS. (A) prp8 alleles identified in different screens were tested for effects on reporters containing various mutations at the 5′SS, BP, or 3′SS. Each column shows growth on the concentration of copper that is limiting for each reporter in a wild-type PRP8 strain. Equal numbers of log phase cells for each mutant strain were spotted onto the same copper containing plate. The prp8 alleles do not confer strong effects on growth when the ACT1–CUP1 reporter is wild type, (C-133, the exception, is temperature sensitive). Not visible, some but not all of the alleles from each of the screens were observed to confer a mild enhancement of copper resistance to the wild-type ACT1–CUP1 reporter. Because some Pyr alleles display this puzzling effect, it does not appear to correlate with the 5′SS and 3′SS suppression phenotype. (B) Phenotypes for prp8 alleles identified in three independent screens are summarized. (+) Intron mutations that are suppressed; (−) intron mutations that are not suppressed. Alleles identified through independent screens for 5′SS position 2 and 3′SS YAG suppression display the same splice site suppression phenotype.

The exon ligation defect of 5′SS U2A and U2G splicing is suppressed. (A) Primer extension analysis of ACT1–CUP1 RNA splicing. Products generated from precursor, mature, lariat-intermediate, and U14 control species are denoted at left of the gel. Other bands are prominent primer extension stops derived from the longer precursor species. (Right) Splicing of a wild-type ACT1–CUP1 reporter is shown, to highlight the splicing defect conferred by the U2A mutation and the modest extent of suppression by prp8 alleles. Note that this experiment used less total RNA, as indicated by the reduced U14 levels. (B) The efficiency of the second step is estimated by calculating the ratio of levels of mature spliced ACT1–CUP1 RNA to the lariat intermediate species (Pikielny and Rosbash 1985; Fouser and Friesen 1986). The values were determined through PhosphorImager analysis of triplicate samples, with the exception of C-133 (for U2A). The near background levels of lariat intermediate in this strain resulted in large deviations for the (Mat/Lar int.) measurement. Shown is the lowest estimate for this ratio. (C) The total splicing efficiency is estimated by the ratio of levels of mature spliced ACT1–CUP1 RNA to unspliced precursor ACT1–CUP1 RNA.

Splice site suppressor alleles of prp8 suppress the dominant-negative phenotype of mutations in U6 A51. (A) Growth (at 30°C) conferred by each prp8 allele in strains containing wild-type or A51C mutant U6 on a plasmid, in addition to a wild-type chromosomal copy of U6 snRNA. Equal numbers of log phase cells were spotted onto medium lacking histidine to require retention of the HIS3-marked plasmid containing dominant-negative U6. (B) The exon ligation defect conferred by dominant-negative mutations in U6 A51 is partially suppressed by prp8 alleles. Primer extension analysis of wild-type ACT1–CUP1 reporter RNA was conducted in triplicate for wild-type PRP8 and C-122 strains. The efficiency of the exon ligation step of splicing was estimated as described in Fig. 5. (C) prp8 suppressor allele C-122 allows for expression and association of U6 A51C. Strains containing mutant or wild-type prp8, and a shorter, pseudo-wild-type version of U6 snRNA (pwtU6), were transformed with an additional plasmid containing full-length U6*: either wild-type or A51C. Total RNA and RNAs that coimmunoprecipitation with polyclonal α-Prp8 were resolved on a denaturing gel and subjected to Northern analysis with an oligo probe that hybidizes to both pwtU6 and U6*(Madhani et al. 1990). The polyclonal antibodies immunoprecipitate Prp8 with a very low efficiency, (data not shown), but the critical comparison is the ratio of mutant U6*/pwtU6. This ratio is not significantly different between C-122 (mut) and wild-type (wt) PRP8. (Lane Pre) Background levels of RNA precipitated by preimmune sera.

Hypothesized interaction of RNA residues suppressed by prp8 extends the juxtaposition of conserved residues at the 5′ and 3′ ends of the intron. (A,B) The previously implicated non-Watson–Crick base-pairing interaction between the first and last residue of the intron (*), in addition to Watson–Crick (−) and cross-linking (lightning bolt) interactions between the 5′SS and U6 snRNA, equally favor an alignment of the 5′ and 3′ ends of the intron in which additional residues are juxtaposed (A), or an alignment in which they are not (B). Residues that are critical for the second step are denoted in outline. The 3′ hydroxyl of exon 1 and the 3′SS phosphate, reactants for the exon ligation chemical step are colored green. (C) Prp8 could function in conjunction with RNA interactions at the spliceosomal active site for the second step. The known and hypothesized (asterisks) RNA interactions could align the 5′ and 3′ ends of the intron, in addition to the 5′ and 3′ exons, to position the reactants (green) for exon ligation catalysis. Interactions between the invariant loop of U5 snRNA and the 5′ and 3′ exons are shown in blue.