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. 1998 Apr;18(4):2055-66.
doi: 10.1128/MCB.18.4.2055.

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing

D Xu  1 , D J Field, S J Tang, A Moris, B P Bobechko, J D Friesen
Affiliations

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing

D Xu et al. Mol Cell Biol. 1998 Apr.

Abstract

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.

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Figures

FIG. 1
FIG. 1
(A) Yeast U2 snRNA. The top diagram shows the structure of yeast U2 snRNA with the 5′-end region, including stems I and II. BP int, the region that interacts through base pairing with the branchpoint (BP) site in pre-mRNA. The bottom diagram represents U2-U6 snRNA interactions (intermolecular helices Ia, Ib, and II), illustrated in the context of BP-U2 (BP) and 5′-splice-site–U6 (5′ SS) interactions, and alignment of exons by U5 snRNA (Alignment). Nucleotides involved directly in the transesterification reaction are circled. Dots indicate the U2-U6 snRNA helix II region that is mutated in the 11-nt substitution (Sub.) mutation of U2 snRNA. The 11-nt substitution (12) used in the genetic screen and other U2 snRNA mutations used to test allele specificity are shown under U2 snRNA in the context of U2-U6 snRNA interactions. (B) Growth phenotypes of yeast strains carrying the U2 snRNA mutations shown in panel A. (C) Yeast strain used in the genetic screen. The strain (12) contains a chromosomal deletion of the U2 snRNA gene (SNR20), which was replaced with HIS3 (SNR20Δ::HIS3), and two plasmids carrying wt SNR20 (URA3 CEN-ARS, i.e., a maintenance plasmid) and mutant snr20-11nt (TRP1 CEN-ARS). Following EMS mutagenesis, cells harboring extragenic mutants (slt’s) that became synthetically lethal with the mutant U2 snRNA were also sensitive to 5-FOA. (D) Growth phenotypes of six slt mutants. In addition to conferring synthetic lethality, these slt mutants all confer growth defects at various elevated temperatures. Shown are the 2-day growth phenotypes of the slt strains obtained following a series of back-crosses with a wt strain (carrying SNR20) grown on yeast extract-peptone-dextrose medium. Note that the slt15, slt16, and slt22 strains all grow slowly at 30°C.
FIG. 1
FIG. 1
(A) Yeast U2 snRNA. The top diagram shows the structure of yeast U2 snRNA with the 5′-end region, including stems I and II. BP int, the region that interacts through base pairing with the branchpoint (BP) site in pre-mRNA. The bottom diagram represents U2-U6 snRNA interactions (intermolecular helices Ia, Ib, and II), illustrated in the context of BP-U2 (BP) and 5′-splice-site–U6 (5′ SS) interactions, and alignment of exons by U5 snRNA (Alignment). Nucleotides involved directly in the transesterification reaction are circled. Dots indicate the U2-U6 snRNA helix II region that is mutated in the 11-nt substitution (Sub.) mutation of U2 snRNA. The 11-nt substitution (12) used in the genetic screen and other U2 snRNA mutations used to test allele specificity are shown under U2 snRNA in the context of U2-U6 snRNA interactions. (B) Growth phenotypes of yeast strains carrying the U2 snRNA mutations shown in panel A. (C) Yeast strain used in the genetic screen. The strain (12) contains a chromosomal deletion of the U2 snRNA gene (SNR20), which was replaced with HIS3 (SNR20Δ::HIS3), and two plasmids carrying wt SNR20 (URA3 CEN-ARS, i.e., a maintenance plasmid) and mutant snr20-11nt (TRP1 CEN-ARS). Following EMS mutagenesis, cells harboring extragenic mutants (slt’s) that became synthetically lethal with the mutant U2 snRNA were also sensitive to 5-FOA. (D) Growth phenotypes of six slt mutants. In addition to conferring synthetic lethality, these slt mutants all confer growth defects at various elevated temperatures. Shown are the 2-day growth phenotypes of the slt strains obtained following a series of back-crosses with a wt strain (carrying SNR20) grown on yeast extract-peptone-dextrose medium. Note that the slt15, slt16, and slt22 strains all grow slowly at 30°C.
FIG. 2
FIG. 2
Splicing defects associated with slt mutations. (A) In vitro splicing defects associated with the slt11, slt22, and slt17 mutations. Splicing reactions with 32P-labeled actin pre-mRNA substrate were performed at 25 and 33°C for 20 min with whole-cell extracts prepared from wt, slt11, slt22, and slt17 cells. Precursor, intermediates (free 5′ exon and lariat intron–3′ exon), and final products (5′ exon–3′ exon and lariat intron) of the splicing reaction are indicated between the gels. Arrows in lanes 12 and 14 indicate preferential accumulation of lariat-intron–3′-exon intermediate in slt17 extract. (B) Primer-extension analyses of inhibition of splicing in vivo. The left gels show reduced levels of mature actin RNA in slt15 cells. The right gels show inhibition of pre-U3 splicing in slt16 cells. The two upper bands labeled A and B correspond to pre-U3A and pre-U3B, respectively. Cells were grown at 25°C for several generations and then were shifted to 37°C. Total yeast RNA was isolated before (grown at 25°C) and following a shift to 37°C for the times indicated. Levels of spliced and unspliced RNAs were measured by primer extension with labeled oligonucleotide complementary to the second exon. Precursor and mature RNAs are indicated.
FIG. 3
FIG. 3
Genetic interactions between slt mutations and the U2-U6 snRNA helix II. (A) Representative yeast strain used in the genetic tests. (B) U2 and U6 snRNA mutations used in the genetic tests. The U6 snRNA mutations and their growth phenotypes in a wt (SLT) background are described in reference . (C) Growth for 2 days at 30°C of four slt strains containing various combinations of U2 and U6 snRNA mutations. Mutant U2 snRNA (TRP1-marked) and U6 snRNA (LEU2-marked) plasmids were introduced into the yeast strains shown in panel A. The resultant transformants were grown on 5-FOA-containing selective medium. Note that the slt22-1 mutation confers slow growth at 30°C.
FIG. 4
FIG. 4
Summary of genetic interactions among factors involved in either or both steps of splicing. Thick lines indicate synthetic lethality at all temperatures. Thin lines indicate partial synthetic lethality (Table 4). None of the slt16/smd3-1, slt22-1, and prp2-1 mutations show synthetic lethality with other mutations tested. Genetic interactions between second-step mutations, including prp18 and prp8, have also been described elsewhere (14, 20, 48). Pi, inorganic phosphate.
FIG. 5
FIG. 5
Synthetic lethality of slt mutants with loop 1 mutations of U5 snRNA. (A) Yeast strains carrying slt mutations and a chromosomal deletion of the U5 snRNA gene (SNR7Δ::HIS3), with SNR7 on a URA3 CEN-ARS plasmid, were transformed with wt and mutant U5 snRNA plasmids (LEU2 CEN-ARS). (B) Results of 5-day growth at 30°C of the resultant transformants on medium containing 5-FOA. Note that slt22-1 and U5 snRNA double mutants grew significantly slower than strains carrying either mutation alone.
FIG. 6
FIG. 6
Genetic interactions (synthetic lethality and suppression) between substitutions at the G21 position in U2 snRNA and loop 1 mutations of U5 snRNA. (A) Yeast strain containing both SNR20Δ::HIS3 and SNR7Δ::HIS3. Mutant U2 snRNA (TRP1 CEN-ARS) and U5 snRNA (LEU2 CEN-ARS) plasmids were introduced into this strain to test for genetic interactions. The resultant transformants were first grown on 5-FOA-containing medium at both 25 and 30°C. Cells containing snr20-G21C and snr7-U97C/U99C failed to grow on this medium; i.e., they are synthetically lethal. Other 5-FOA resistant cells were then grown on selective medium at the temperatures indicated. (B) Four-day growth at 25 and 30°C of U5 snRNA-wt, -U98A, -U97C/U99C in combination with U2 snRNA-wt, -G21A, -G21C, and -G21U in the absence of a maintenance plasmid. Note that U2-G21C is synthetically lethal with U5-U97C/U99C. (C) Summary of genetic interactions between U2 and U5 snRNAs and the locations of U2 and U5 snRNA mutations in the context of other demonstrated RNA-RNA interactions in the spliceosome. Positions 97, 98, and 99 in U5 snRNA loop 1 are enclosed in filled squares. Filled circles indicate mutations at positions in U2 snRNA that are synthetically lethal with the U5 snRNA mutations tested (see Table 5 for a summary). G21 of U2 snRNA is enclosed in a filled square; mutations at this position were able to suppress the U5 snRNA mutations tested. The U2 snRNA part of the U2-U6 snRNA helix II is shaded. Substitutions in this region (i.e., the 11-nt substitution) cause synthetic lethality with slt mutations but not with U5 snRNA mutations. Lines from U23 and A30 in U2 snRNA to exon 2 indicate site-specific cross-linking (33).
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