INTRODUCTION

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Precursor-mRNA (pre-mRNA) splicing takes place in the spliceosome through a two-step transesterification reaction. At least 40 splicing factors have been identified by genetic means in the yeast Saccharomyces cerevisiae. Most have been implicated in specific steps of the splicing pathway (31, 41). During the process of spliceosome assembly, small nuclear RNAs (snRNAs) and the pre-mRNA substrate, in association with protein factors, undergo extensive conformational changes which establish RNA-RNA interactions that are important for both splicing reactions. Among these factors are the DExD or DExH proteins: RNA-dependent ATPases (possibly RNA helicases), which include Prp2p, Prp5p, Prp16p, Prp22p, Prp28p (26, 31), Prp43p (2), and Slt22p (also called Brr2p) (22, 34, 53). Their functions are essential for the formation and maintenance of RNA-RNA interactions in the spliceosome.

With few exceptions (28, 29, 36), Watson-Crick base pairing is important for most RNA-RNA interactions in the formation of the spliceosome (26). In particular, intermolecular base-pairing interactions that occur between U2 and U6 snRNAs, forming helices I and II (Fig. 1A), are likely to be involved in bringing the 5' splice site (through a U6-5'-splice-site interaction [21, 23, 42]) and the branchpoint site (through a U2-branchpoint interaction [37]) into proximity. This is necessary for the first-step reaction (Fig. 1A). Residues in both U2 and U6 snRNAs that are important for either or both steps of splicing (11, 30) form part of these interactions or lie nearby. This finding has led to the suggestion that these RNA structures may form the so-called active center for catalysis of the splicing reactions (27, 30). The helix II portion of human U6 snRNA has also been shown to influence dissociation of the U4-U6 snRNA duplex in vitro, perhaps by stabilizing it (5).

View larger version (37K): [in this window] [in a new window]   View larger version (52K): [in this window] [in a new window]   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 (SNR20::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.

The highly conserved loop 1 region of U5 snRNA is important for the juxtaposition of the two exons, which is essential for the second step of splicing (35). However, canonical base pairing is not obviously involved in this interaction, suggesting that additional factors are required for its establishment and maintenance. In fact, two splicing factors, Prp8p (45, 47, 48) and Slu7p (6, 13), have been implicated in the alignment of the two exons and/or in 3'-splice-site selection. In addition, a functional role for U2 snRNA in the alignment of the two exons has been suggested by site-specific cross-linking between the first nucleotide of the 3' exon and the U23 and A30 nucleotides of U2 snRNA (33). Despite the available information, it is not clear how different RNA structures in the active center are coordinated, either spatially or temporally, prior to and after the first splicing step.

The yeast U2 snRNA contains all conserved elements found in other eukaryotic U2 snRNAs, despite its unusually large size (approximately 1,200 nucleotides [nt]) (Fig. 1A). The stem II region of U2 snRNA is important for recognition of the branchpoint site and association of the U2 snRNP with the pre-mRNA. Several factors, including Prp5p (a DEAD-box protein), SF3a (Prp9p Prp11p Prp21p), and SF3b, have been implicated in this function (40, 51, 54). In contrast, relatively little is known about the factors involved in subsequent steps of spliceosomal function during which the stem I region of U2 snRNA undergoes extensive conformational rearrangements to form the U2-U6 snRNA interactions (see above and Fig. 1A).

We have devised a synthetic-lethality genetic screen to search for such factors. In this screen, a mutant U2 snRNA carrying an 11-nt substitution in the stem I region (12) was used as the starting mutation. Although this mutation can potentially perturb the U2-U6 snRNA helix II interaction (Fig. 1A), it confers only a mild growth defect, which is likely due to functional redundancy of the helix Ib and helix II regions in U2-U6 snRNA (12). Our genetic screen yielded six slt (stands for synthetic lethality with U2) mutants. We have characterized and cloned all the SLT genes, two of which encode new splicing factors: Slt11p (a possible RNA-binding protein), Slt22p (an RNA-dependent ATPase [53]), and four previously characterized factors, Slt15p (previously named Prp17p [Slt15p/Prp17p]), Slt16p/Smd3p, Slt17p/Slu7p, and Slt21p/Prp8p. These factors are required for either or both steps of splicing. Our genetic and biochemical characterizations suggest that the functions of these factors and of U2 snRNA may overlap that of invariant loop 1 of U5 snRNA in the alignment of the two exons in the spliceosome.

	MATERIALS AND METHODS

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Yeast strains and plasmids. All yeast strains used in this study were derived from W303-1A or -1B (MATa or MAT ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100). The original strain used in the genetic screen, containing a chromosomal deletion of SNR20 (the U2 snRNA gene), has been described previously (12). A new SNR20 disruption was made by deleting the ClaI-HpaI region of ~810 nt containing the 5'-end half of the U2 snRNA gene (~750 nt) and by replacing it with the yeast HIS3 gene. The wild-type (wt) SNR20 gene was carried on pRS316 (URA3 CEN-ARS). Two resultant haploids, YDX2299A (MATa) and YDX22100A (MAT), were used in the subsequent genetic experiments. Chromosomal deletion of SNR6 (the U6 snRNA gene) was constructed by deleting the entire coding region and replacing it with the yeast HIS3 gene. Two haploid SNR6 strains, YXU91 (MAT) and YXU92 (MATa), were obtained. Strains containing slt11, slt17, slt21, and slt22 mutations in the SNR20 background were crossed to both SNR6 strains in order to generate haploid strains carrying double deletions as well as slt mutations. SNR20 and SNR6 were carried on the same maintenance plasmid. These strains were used to test genetic interaction among slt, U2, and U6 mutations. U6 snRNA mutations (3-nt substitutions near the 3' end: designated a, b, c, and d) have been described (12).

EMS mutagenesis, screening, and genetic characterization. In the initial genetic screen, a yeast strain with a chromosomal deletion of SNR20 (12) containing SNR20 and snr20-11nt carried on URA3 and TRP1 plasmids, respectively, was subject to ethyl methanesulfonate (EMS) mutagenesis to yield a survival rate of 10 to 20%. The surviving cells were screened for sensitivity to 5-fluoroorotic acid (5-FOA) at 30°C. This indicates dependence of viability on SNR20 (carried on a URA3 plasmid), which reflects the lethality generated by snr20-11nt in combination with extragenic mutations (i.e., synthetic lethality) (Fig. 1C). In order to eliminate artifacts (e.g., mutations which affect the uracil pathway), the snr20-11nt/TRP1 plasmids in 5-FOA-sensitive cells were replaced by another SNR20 plasmid. The resultant cells that were sensitive to 5-FOA were discarded. A scheme for genetic characterization was designed to ensure that the slt mutants were phenotypically and genetically appropriate (they should have arisen from mutation at a single locus which also confers a recessive growth defect). Briefly, the temperature-sensitive phenotype of each original slt strain was first segregated into the chromosomal SNR20 background by crossing the slt strain with the wt strain (W303-1A). The resulting temperature-sensitive haploid was then back-crossed with the wt strain at least three times to ensure that the temperature sensitivity phenotype was due to mutation at a single locus. The final temperature-sensitive haploid was crossed with the newly constructed SNR20 deletion strain (YDX2299A or YDX22100A). Synthetic lethality was then tested in strains containing both the temperature sensitivity mutation and SNR20 derived from the above-described cross. These strains were also used to generate the slt SNR20 plus SNR6 strains described above.

Splicing extract preparation and in vitro splicing. Yeast whole-cell extracts were prepared from wt and slt cells according to the method of Lin et al. (24) with modifications. The substrate³²P-labeled yeast pre-actin RNAwas synthesized by runoff transcription with T7 RNA polymerase and ³²P[UTP] as described previously (24). For in vitro splicing assays, equal volumes (10 µl) of whole-cell extract and buffer component, containing labeled substrate, were mixed and incubated at the temperatures indicated in the figures. Splicing intermediates and products were resolved in a 5% polyacrylamide gel.

RNA isolation and primer extension. All slt mutant strains used for RNA analysis contained SNR20 on the chromosome (see above). Total yeast RNA isolation and primer extension were performed as described previously (17).

Cloning by complementation. In order to clone the wt SLT genes, a YCp50-borne yeast genomic DNA library, CENBANK A (38), was introduced into slt cells and the transformants were selected for temperature resistance at 37°C. Plasmids containing complementing genomic DNA fragments were recovered from the positives. A minilibrary approach was used to define the minimal complementing regions. The original complementing plasmid was first digested with a variety of restriction enzymes, and the resulting fragments were ligated to another vector with a different selective marker. Total plasmid DNA prepared from these mini-libraries was introduced into the slt strains, followed by selection for full complementation. The overlapping region in plasmids rescued from different mini-libraries contain slt-complementing open reading frames. Nucleotide sequences of both ends of these fragments were determined and then used to search the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) for a match. DNA sequences of complementing regions were retrieved, and fragments containing only single open reading frames were tested for complementation of both growth defect and synthetic lethality.

Genetic analysis of synthetic lethality. In order to examine potential synthetic lethality of two mutations, a heterozygous diploid was first generated from two parental slt (slu or prp) haploid strains and was then subjected to sporulation and tetrad dissection. If haploids containing both mutations are viable, three types of tetrads should be obtained: parental ditype (PD; each spore inherits either of the original mutations), nonparental ditype (NPD; two spores inherit both mutations, while the other two inherit neither mutation, i.e., it has the wt phenotype), and tetratype (T; two spores inherit either of the original mutations, one inheriting both mutations and the remaining one inheriting neither mutation, i.e., it has the wt phenotype). However, if the combination of both mutations is lethal, only PD tetrads can be obtained from such a diploid. T tetrads should contain only three viable spores (one wt spore and two spores with either mutation), and NPD tetrads should contain only two wt spores; the lack of complete (i.e., with four viable spores) NPD and T tetrads indicates that the two mutations in question are synthetically lethal. In most cases, at least 20 scoreable tetrads from each heterozygous diploid were analyzed phenotypically to determine synthetic lethality.

	RESULTS

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Genetic screening, characterization, and molecular cloning of SLT genes. The U2 snRNA mutation (snr20-11nt) used in the search for synthetic-lethality mutants contains an 11-nt substitution in the stem I region (Fig. 1A) (12). Although such a substitution may potentially disrupt the proposed U2 snRNA stem structure and perturb the helix II interaction of U2-U6 snRNA, it confers only a mild growth defect at both 37 and 16°C (12) (Fig. 1B). Cells containing the wt U2 snRNA gene (SNR20) carried on a URA3-marked plasmid and snr20-11nt on a TRP1-marked plasmid were mutagenized with EMS. Surviving cells were screened for extragenic mutations that failed to allow growth at 30°C in the absence of SNR20 (as indicated by sensitivity to 5-FOA). These extragenic mutations result from lethality generated by snr20-11nt in combination with a second mutation (Fig. 1C), i.e., they produce synthetic lethality, which suggests functional interaction between the two gene products. These mutations were designated slt mutations (for synthetic lethality with U2 snRNA).

View larger version (52K): [in this window] [in a new window]   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.

SLT11. SLT11, a new splicing-related gene, corresponds to YBR065c. It encodes a 41-kDa protein of 364 amino acids. Two putative zinc fingers (CX₂CX₁₇CX₂C and CX₂CX₆CX₂C) are present in the N-terminal region, while the central region (amino acids 151 to 299) shows homology (27% identity and 40% similarity) to yeast ribosomal protein L25. The C terminus (approximately 30 amino acids) of Slt11p is highly charged due to the presence of blocks of lysine residues. The original slt11-1 mutation blocks splicing prior to the first step (Fig. 2A, lane 5) without an apparent effect on spliceosome assembly (unpublished data), suggesting that Slt11p exerts its function immediately prior to the first-step splicing reaction.

SLT15/SLU4/PRP17. SLT15/SLU4/PRP17 was first isolated in a genetic screen for temperature sensitivity mutations that also affect pre-mRNA splicing (prp17 [49]) and in another screen independently (slu4 [14]). The encoded protein contains WD repeats, which are also present in another splicing factor, Prp4p (4, 17). Prp17p is involved exclusively in the second step of splicing (20). The new allele is named prp17-100. The primary defect of the slt15/prp17-100 mutation was associated with the reduction of mature RNA, and no preferential accumulation of unspliced precursor was observed (Fig. 2B, lanes 6 through 8), suggesting that the mutation affects the stability of mature RNA and/or, more likely, the second step of splicing.

SLT17/SLU7. SLT17/SLU7 encodes a second-step splicing factor (6, 13, 20) (Fig. 2A, lanes 11 through 14). The gene product contains a so-called zinc knuckle that is similar to one in retroviral nucleocapsid protein and that is required for RNA-protein interaction (13). The slt17 allele is renamed slu7-100. Both slu4-1/prp17-2 and slu7-1 were isolated in a genetic screen for splicing mutations that are synthetically lethal with mutations in invariant loop 1 of U5 snRNA (14), a region that has been implicated in alignment of the two exons in the second step of splicing (35). However, Slu7p and Prp17p exert their functions at different steps, with respect to that of hydrolysis of ATP by Prp16p (20), a putative RNA helicase, whose function is required for the second step (7, 8, 43).

SLT16/SMD3. SLT16/SMD3 encodes a yeast core Sm protein with homology to human SmD3 (39). slt16 is the first reported temperature-sensitive allele of this gene and is renamed smd3-1. The protein is associated with all five spliceosomal snRNAs and is thought to be involved in the biogenesis of snRNPs. In an extract depleted of Smd3p, splicing is blocked before or at the first step (39). A similar in vivo splicing defect was observed for the slt16/smd3-1 mutation (Fig. 2B, lanes 12 through 14). The human SmD3 protein has been shown to be cross-linked to the 5' splice site in a site-specific manner (25, 52). To our knowledge, this study is the first time that an Sm protein has been shown genetically to be important for the function of a particular snRNA in splicing. However, it remains to be determined whether this genetic interaction between Smd3p and U2 snRNA is direct (affects the function of U2 snRNA) or indirect (affects the stability of snRNAs).

Genetic interactions of slt, slu, and prp mutations with U2 snRNA. We determined whether the phenotypes of the slt mutations are specific for the 11-nt substitution in the stem I region of U2 snRNA, with which they were originally identified. We included in our genetic analysis three sets of U2 snRNA mutations that lie in regions that are involved in U2-U6 snRNA helices: helix Ia (G26A and A27C), helix Ib (substitutions at G21), and helix II (11- and 9-nt substitutions) (Fig. 1A). In addition to the six slt mutations identified in this study, four slu (14) and four prp mutations were also included (Table 2).

Genetic interactions of slt mutations with U6 mutations in U2-U6 snRNA helix II. The original 11-nt substitution in U2 snRNA used in our genetic screen has the potential to perturb U2-U6 snRNA helix II (12). Consequently, we determined whether this genetic interaction extended to nucleotides on the U6 snRNA side of helix II. Yeast strains which contain slt mutations in combination with double deletions of SNR20 and SNR6 were constructed (Fig. 3A). These were used to determine whether specific U6 snRNA mutations (12) were synthetically lethal with the slt mutations and whether partial restoration of a mutationally disrupted helix II could suppress the synthetic lethality that is observed with the original slt mutations. (Note that we were unable to determine whether a corresponding 9-nt U6 snRNA substitution conferred synthetic lethality, since that mutation is lethal [12].) Two slt mutations that encoded new splicing factors (slt11-1 and slt22-1) and two that encoded existing ones (slt17/slu7-100 and slt21/prp8-21) were tested.

View larger version (35K): [in this window] [in a new window]   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 12. (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.

Genetic interactions among slt and slu mutations. The fact that the slt mutations are all synthetically lethal with certain U2 snRNA mutations suggested that at least some of them might form a functional unit(s). We assessed this possibility by testing the synthetic lethality of pairs of slt mutations, as well as of selected prp mutations, by attempting to create double-mutant haploids (Table 3 and Materials and Methods). Failure to obtain such haploids indicates that the two mutations in question are synthetically lethal. The results of these experiments are summarized in Tables 3 and 4 and Fig. 4.

View larger version (34K): [in this window] [in a new window]   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.

Genetic interactions among slt and U5 snRNA mutations. All of our slt mutations were isolated as synthetically lethal with a U2 snRNA mutation, yet two of them (slt15 and slt17) are in the same genes (slu4 and slu7, respectively) as mutations that were identified originally as being synthetically lethal with U5 snRNA mutations (14). Furthermore, three (possibly four) Slt factors may act in functional groups (see above), which suggests the possibility of an interaction involving some of the Slt factors and U5 snRNA.

View larger version (52K): [in this window] [in a new window]   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 (SNR7::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.

Genetic interactions between U2 and U5 snRNAs. The data presented above indicate that members of a group of splicing factors, which were identified originally on the basis of their genetic interaction with U2 snRNA, also interact with one another and with U5 snRNA. It is possible that these factors act together in functions related to both U2 and U5 snRNAs, such as coordination of the two steps of splicing and/or tethering of the two exons following the first splicing step (35). This suggests that there may be genetic interactions between U2 and U5 snRNAs. The two U5 snRNA mutations used in the genetic screen for slu mutants (14), U98A/U99C, and a series of U2 snRNA mutations were tested for genetic interaction with the yeast strain shown in Fig. 6A.

View larger version (33K): [in this window] [in a new window]   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 SNR20::HIS3 and SNR7::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).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The genetic screen described here has identified six yeast splicing factors (called slt factors). Mutations in genes encoding these factors become synthetically lethal with mutations in the stem I region of U2 snRNA (corresponding to helix II of the U2-U6 snRNA composite structure). Each of these splicing factors affects either or both steps of splicing. One of these factors, Slt22p, is likely to be involved with U2-U6 snRNA helix II (53). Three factors, Slt15p/Prp17p, Slt17p/Slu7p, and Slt21p/Prp8p, are linked to the tethering of U5 snRNA loop 1 (14, 45). On the basis of genetic interactions among some members of this group of factors and other prp mutations, we suggest that Slt11p, Prp17p, Slu7p, Prp16p, and Prp8p form two overlapping functional units. Four slt mutations in this group are also synthetically lethal with mutations in U5 snRNA loop 1. From this we conclude that these factors, including a new one, Slt11p, may be involved in the functions of both U2 and U5 snRNAs, perhaps in alignment of the two exons in the yeast spliceosome and/or coordination of the two steps of splicing. Genetic tests indicated an interaction between mutations in loop 1 of U5 snRNA and those near the 5' end of U2 snRNA. Given the role of U5 snRNA loop 1 in the tethering of exons (35), it may be that U2 snRNA also plays a role in this function. Our findings suggest a mechanism for coupling and coordination of the two steps of splicing.

The functions of U2-U6 snRNA helix I and helix II regions. During maturation of the spliceosome, snRNAs and the pre-mRNA substrate undergo conformational changes in order to juxtapose the splice sites and to form structures that are important for the subsequent steps of splicing (26). For both helix I and helix II of U2-U6 snRNA, there is evidence that the U6 snRNA components may have functions that are independent of their interaction in the helical structures. For example, mutations in the U6 snRNA components of yeast helix II are lethal and cannot be suppressed by base-compensatory mutations in the corresponding U2 snRNA portion of U2-U6 snRNA (12). Similar genetic asymmetry was also observed for nucleotides involved in the helix Ib interaction (11, 27, 28). Indeed, the 5' end of U2 snRNA itself may be important for the second step of splicing. The genetic interactions of both Slt17p/Slu7p and Slt21p/Prp8p with U2 snRNA are specific to the U2 portion of U2-U6 snRNA helix II (Fig. 3). This observation differentiates the function of the 5'-end region of U2 snRNA from its role in U2-U6 snRNA helix II.

Interactions between the U2 and U5 snRNPs. The genes encoding the Slt factors were isolated on the basis of genetic interaction with U2 snRNA, yet four of them are related functionally to U5 snRNA. This finding suggests that the U2 snRNA portion of U2-U6 snRNA helix II (the 5'-end region), following resolution of this helix by Slt22p, may interact with U5 snRNA. Three Slt factors are related to the tethering function of U5 snRNA loop 1. Slt21p/Prp8p, which has been shown to be cross-linked to both the 5' and 3' splice sites in human (52) and yeast (45, 47, 48), may act to stabilize the exon-U5 snRNA loop 1 interaction (45). This factor is also involved in the recognition of the polypyrimidine tract (46, 47) and 3'-splice-site selection (46). The other two factors, Prp17p and Slu7p, are linked genetically to the function of U5 snRNA loop 1 (14) and are in close contact with the 3' splice site (1, 6, 13, 20, 48).

Interactions among Slt factors: coordination of the two steps of splicing. Four slt mutations, slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21, show pairwise synthetic lethality with each other and with prp16-1 (Fig. 4). prp16-1 was identified initially as a suppressor of a mutation at the branchpoint site; this mutation resulted in a second-step block due to the use of a cryptic branchpoint site (7). McPheeters (29) has shown genetic suppression of nonadenosine branchpoints by mutations in two regions of U6 snRNA that are either part of U2-U6 snRNA helix Ia (U57) or of the intramolecular stem-loop adjacent to helix Ib (nt 63 to 65, 71, and 82 to 84) and that an allele of prp16, prp16-302, is synthetically lethal with one of these U6 snRNA suppressors, U57C (29). Our results demonstrate that prp16-1 is synthetically lethal with U2 substitutions in the U2-U6 snRNA helix Ia region (G26A).