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• W2112654577 abstract "Article2 November 1998free access The yeast U2A′/U2B″ complex is required for pre-spliceosome formation Friederike Caspary Friederike Caspary EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Bertrand Séraphin Corresponding Author Bertrand Séraphin EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Friederike Caspary Friederike Caspary EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Bertrand Séraphin Corresponding Author Bertrand Séraphin EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Author Information Friederike Caspary1 and Bertrand Séraphin 1 1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6348-6358https://doi.org/10.1093/emboj/17.21.6348 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human U2 snRNP contains two specific proteins, U2A′ and U2B″, that interact with U2 snRNA stem–loop IV. In Saccharomyces cerevisiae, only the counterpart of human U2B″, Yib9p, has been identified. Database searches revealed a gene potentially coding for a protein with striking similarities to human U2A′, henceforth called LEA1 (looks exceptionally like U2A′). We demonstrate that Lea1p is a specific component of the yeast U2 snRNP. In addition, we show that Lea1p interacts directly with Yib9p. In vivo association of Lea1p with U2 snRNA requires Yib9p. Reciprocally, Yib9p binds to the U2 snRNA only in the presence of Lea1p in vivo, even though it has been previously shown to associate on its own with the U2 snRNA stem–loop IV in vitro. Strains lacking LEA1 and/or YIB9 grow slowly, are temperature sensitive and contain reduced levels of U2 snRNA. Pre-mRNA splicing is strongly impaired in these cells. In vitro studies demonstrate that spliceosome assembly is blocked prior to addition of U2 snRNP. This phenotype can be rescued partially, but specifically, by addition of the corresponding recombinant protein(s). This demonstrates a specific role for the yeast U2 snRNP specific proteins during formation of the pre-spliceosome. Introduction Pre-mRNA splicing occurs through two transesterification reactions. For nuclear pre-mRNA splicing these reactions take place in a large RNA–protein complex named the spliceosome (Moore et al., 1993). The spliceosome is assembled onto the pre-mRNA by the stepwise addition of small nuclear ribonucleoproteins (snRNPs), which consist of a small nuclear RNA (snRNA) and proteins. In addition, several non-snRNP splicing factors are required for spliceosome assembly and interact transiently with the spliceosome (reviewed in Moore et al., 1993; Krämer, 1996; Will and Lührmann, 1997; Staley and Guthrie, 1998). The ordered pathway of spliceosome assembly has been dissected by following the fate of synthetic RNA substrates in vitro. This process appears to be essentially conserved in different species. First, the U1 snRNP recognizes the 5′ splice site through base pairing with the 5′ end of the U1 snRNA (Rosbash and Séraphin, 1991) leading to the formation of commitment complex 1 in yeast (CC1) (Séraphin and Rosbash, 1989, 1991). A second form of yeast commitment complex (CC2) is then formed through joining of at least two non-snRNP splicing factors, BBP/SF1 (branchpoint binding protein/SF1; Krämer, 1992; Abovich and Rosbash, 1997) and Mud2p/U2AF65 (Abovich et al., 1994), that recognize the 3′ part of the intron. The E complex is likely to be the counterpart of the yeast commitment complexes in metazoans (Michaud and Reed, 1991). These early complexes are the substrates for the ATP-dependent addition of U2 snRNP that leads to formation of the pre-spliceosome. U4/U6·U5 tri-snRNP joins this complex to form the mature spliceosome. The U2 snRNP plays an essential role in spliceosome assembly with U2 snRNA base-pairing with both the pre-mRNA branchpoint sequence and U6 snRNA (Moore et al., 1993). The human U2 snRNP contains two sets of proteins. A group of seven or eight Sm proteins is shared with other snRNPs including U1, U4 and U5. In addition, the U2 snRNP contains two snRNP specific proteins: U2A′ and U2B″ (Lührmann et al., 1990). Biochemical fractionation of HeLa nuclear extracts revealed the presence of an additional form of U2 snRNP. This species, named 17S U2 snRNP, contains nine additional specific proteins and appears to be unstable at high salt concentration (Lührmann et al., 1990; Behrens et al., 1993a, b; Brosi et al., 1993a). Some of these snRNP specific proteins were shown to associate in two subcomplexes: SF3a and SF3b (Brosi et al., 1993b). SF3a is required for pre-spliceosome formation and is conserved from yeast to man (Behrens et al., 1993a; Bennett and Reed, 1993; Brosi et al., 1993a; Legrain and Chapon, 1993; reviewed in Krämer, 1996). SF3b has been only partly characterized; however, some of its subunits have been shown to play specific and essential roles in spliceosome assembly and splicing (Champion-Arnaud and Reed, 1994; Gozani et al., 1996; Wells et al., 1996; Igel et al., 1998; Wang et al., 1998). Surprisingly, the function of the long-known U2A′ and U2B″ has remained unclear. Cloning and sequencing of the cDNA encoding the human U2B″ protein revealed that its sequence is closely related to the U1A protein, a component of the U1 snRNP (Sillekens et al., 1987). Both proteins contain two RNA recognition motifs (RRM) (reviewed by Burd and Dreyfuss, 1994). The N-terminal RRM motif, which is 75% identical between the two proteins is, together with a small number of flanking amino acids, required for binding their cognate snRNAs (Scherly et al., 1990; Bentley and Keene, 1991; Oubridge et al., 1994). The function of the C-terminal RRM, which is the most conserved (86% identity), is still not well understood (Tang et al., 1996). U1A binds to stem–loop II of U1 snRNA while U2B″ interacts with stem–loop IV of U2 snRNA. These two RNA sequences are similar in structure and sequence. However, whereas U1A binds to its RNA target on its own, U2B″ requires the presence of U2A′ for RNA binding (Scherly et al., 1990; Bentley and Keene, 1991). Orthologues of the human U1A and U2B″ proteins have been identified in several eukaryotic species (Polycarpou-Schwarz et al., 1996). Interestingly, the D25/SNF protein of Drosophila melanogaster fulfills the function of both U1A and U2B″, and appears to be a hybrid protein. D25/SNF binds to U1 snRNA without an additional factor, but it still requires co-factors (e.g., human U2A′) to interact with U2 snRNA in vitro (Polycarpou-Schwarz et al., 1996). The human U2A′, which is unrelated in sequence to U1A or U2B″ (Sillekens et al., 1989), is a member of the leucine-rich protein family (Hofsteenge et al., 1988). Members of this family contain tandem repetitions of a motif rich in leucine residues folding in a regular structure that has been proposed to be involved in protein–protein interactions (Kobe and Deisenhofer, 1993). There are six such repeats in human U2A′, located at the N-terminus of the protein, followed by a region with no obvious sequence characteristics. To date, a single orthologue of the human U2A′ protein has been characterized in Trypanosoma brucei. It shows 31% identity and 57% similarity to its human counterpart, the homology being concentrated to its leucine-rich repeats which are located in the N-terminal region (Cross et al., 1993). Although the Trypanosoma U2B″ homologue has not yet been described, it is known that interaction of the Trypanosoma U2A′ with loop IV sequence of U2 snRNA requires additional proteins (Cross et al., 1993). With the completion of the yeast genome sequence, it has become possible to identify (putative) homologues of human splicing factors using sensitive database searches. This allowed for the identification of yeast Sm proteins (e.g. Séraphin, 1995) as well as some snRNP specific subunits (e.g. U1-70K; Smith and Barrell, 1991). Yib9p, the yeast homologue of human U2B″, was identified in this way as well as through a genetic screen (Voss et al., 1995; Polycarpou-Schwarz et al., 1996; Tang et al., 1996). Yib9p harbors only one RRM. Interestingly, it binds to the yeast U2 snRNA stem–loop IV and the human U1 snRNA stem–loop II in vitro (the target of U1A) but interacts only weakly with human U2 snRNA stem–loop IV in the same assay (Polycarpou-Schwarz et al., 1996; Tang et al., 1996). This might result from the higher similarity of yeast U2 snRNA stem–loop IV with human U1 snRNA stem loop II than with its human counterpart. These in vitro studies also revealed that Yib9p is able to bind yeast U2 snRNA in a sequence-specific manner in the absence of other protein factors (Polycarpou-Schwarz et al., 1996). The addition of human U2A′ and yeast extract did not influence this binding (Tang et al., 1996) suggesting that the Yib9p function is more related to human U1A than human U2B″. These results suggested that the U2A′ function might be missing in yeast. Consistent with this, a large-scale two-hybrid screen using Yib9p as a bait failed to uncover a yeast U2A′ homologue (Fromont-Racine et al., 1997). We have used a database search approach to look for a putative homologue of the human U2A′ in Saccharomyces cerevisiae. This identified an open reading frame coding for a protein with striking sequence similarity to human U2A′ which was therefore named LEA1 (looks exceptionally like U2A′). Here, we demonstrate that the encoded protein, Lea1p, associates with U2 snRNA in the presence of Yib9p to which it binds directly. Lea1p is therefore a true homologue of the human U2A′ protein. Characterization of a LEA1-disruption mutant demonstrates that Lea1p is required for splicing in vivo and suggests that Lea1p and Yib9p have no independent functions. In vitro analyses revealed that these two proteins are specifically required for the transition from CC2 to pre-spliceosome during the splicing process. Results Database search for a yeast homologue of human U2A′ To look for a counterpart of human U2A′ in S.cerevisiae, we searched nucleic acid and protein sequence databases (see Materials and methods) using either the human U2A′ or its Trypanosoma homologue as a probe. This identified an open reading frame located on chromosome XVI as the best candidate for a U2A′ homologue. Pairwise alignment indicated that the yeast protein was 52% similar and 29% identical to human U2A′ (data not shown). The similar sizes of the two proteins (238 and 255 amino acids for yeast and human, respectively) further suggested that they might be counterparts. Detailed analysis of the protein alignment revealed that the sequence homology is mainly located in the N-terminal region of the two proteins and corresponds to six leucine-rich repeats (Figure 1). Similar results were obtained with the Trypanosoma homologue of human U2A′ (Figure 1). It is noteworthy that in database searches with either the human or the Trypanosoma protein, the yeast protein gave a much stronger score than any other yeast polypeptide containing leucine-rich repeats. This indicated that sequence similarity was not solely due to the presence of these repeats. As this putative yeast U2A′ homologue looks exceptionally like U2A′, it was named LEA1. Database searches revealed several other putative human U2A′ homologues in Salmo salar (81% identical to human U2A′) and Arabidopsis thaliana, and two partial, but highly related sequences in the worms Onchocera volvulus and Brugia malayi. Multiple sequence alignment of divergent members of this protein family (i.e. excluding S.salar and B.malayi which are highly related to human and O.volvulus proteins, respectively; Figure 1) indicated that several amino acids were conserved in all proteins. Some of those corresponded to highly conserved residues of the leucine-rich repeats. However, others were found in variable positions of the leucine-rich repeats or upstream of the repeats. The presence of conserved residues in the latter subset of positions strongly suggest that these proteins are homologues rather than unrelated members of the leucine-rich protein family. Figure 1.Sequence alignment of the N-terminal regions of human and trypanosoma U2A′ with putative homologues. Amino acid identities and conserved substitutions are highlighted. The six tandem leucine-rich repeats, defined according to Cross et al. (1993), are indicated by numbered boxes. Download figure Download PowerPoint Lea1p is specifically associated with the U2 snRNA To test whether Lea1p is a component of yeast U2 snRNP, we inserted a cassette coding for two IgG-binding units of the Staphylococcus aureus protein A (Puig et al., 1998) downstream of, and in-frame with, the LEA1-coding sequence. Splicing extracts were prepared from the resultant strain and used in an immunoprecipitation experiment. Extracts from isogenic wild-type and SmB–ProtA tagged strains were used as controls. Western blot analysis revealed that the Lea1–ProtA and SmB–ProtA were present at similar levels in the corresponding input and pellet fractions while they were undetectable in the supernatant fractions (data not shown). This indicated that the proteins were stable for the duration of the experiment and efficiently immunoprecipitated. RNAs extracted from the various fractions were analyzed by primer extension for the presence of the five spliceosomal snRNAs (Figure 2). All snRNAs were efficiently immunoprecipitated by the SmB–ProtA protein (Figure 2, lane 8), consistent with other observations (J.Salgado-Garrido, E.Bragado-Nilsson, S.Kandels-Lewis and B.Séraphin, unpublished results). The Lea1–ProtA fusion specifically coprecipitated the U2 snRNA (Figure 2, lane 9, compare with background in lane 7). Trace amounts of U5 and U6 snRNAs could be detected in the Lea1–ProtA IgG-beads pellet after prolonged exposure. This could be due to the presence of low amounts of splicing complexes in the extract. Approximately 50% of the U2 snRNA present in the starting extract was recovered in the pellet (Figure 2, lanes 6 and 9). This suggests that only a fraction of U2 snRNA is associated with Lea1p. Alternatively, dissociation of the Lea1–ProtA fusion from a fraction of the U2 snRNP might have occurred during extract preparation and/or immunoprecipitation. In any case, these results demonstrate that Lea1p associates directly or indirectly, and specifically, with the U2 snRNA. Figure 2.Coimmunoprecipitation of snRNAs by Lea1–ProtA. Extracts from a control wild-type yeast strain (WT), a strain expressing SmB–ProtA and a strain harboring Lea1–ProtA were immunoprecipitated with IgG–agarose beads. The extracted RNAs of input (lane 1–3), supernatant (lane 4–6) and pellet (lane 7–9) fractions were analyzed by primer extension with specific primers for U1, U2, U4, U5 and U6 snRNAs. Position of the corresponding signals are shown on the left. RNA from five times more extract was used for the pellets relative to the input and supernantant fractions. Download figure Download PowerPoint LEA1 disruptants are temperature-sensitive The LEA1 gene was disrupted by replacing its coding sequence with a Kluyveromyces lactis URA3 gene in a diploid strain. After sporulation, tetrads were dissected. All intact tetrads gave rise to four viable spores at 30°C (data not shown). However, we noticed that two spores per tetrad grew slower (data not shown). These spores carried the URA3 marker indicating that strains lacking LEA1 (Δlea1) were viable but impaired for vegetative growth at 30°C. This result was somewhat surprising because disruption of the YIB9 gene encoding the yeast U2B″ protein was reported to confer no growth phenotype (Tang et al., 1996). This situation might be explained by functional differences between YIB9 and LEA1 or by a difference in the strain backgrounds. We therefore repeated the disruption of the YIB9 gene in our standard strain (Δyib9) and also constructed an isogenic strain carrying both gene disruptions (Δlea1 Δyib9). All strains were viable, allowing us to compare their growth behavior. The strains were grown in liquid media at different temperatures and cell density was measured at various time points. Compared with an isogenic wild-type strain, the three mutant strains grew with the same reduced rate at 30°C (1.3-fold reduction; data not shown). This phenotype was more pronounced at 16°C, where the mutant strains grew at half the rate of the wild-type strain (data not shown). At 37°C the three mutant strains stopped growing after ∼23 h of incubation (Figure 3A). Similar phenotypes were observed on solid media (data not shown). These results indicate that disruption of the LEA1 gene is not lethal but confers a slow growth phenotype, particularly at low temperatures, as well as a thermosensitive lethal phenotype. Similar phenotypes were observed following disruption of the YIB9 gene. Interestingly, the growth of the strain disrupted for both the LEA1 and YIB9 gene was identical to the growth of the two single disruptants (Figure 3A) suggesting that the two proteins have no independent function. Figure 3.Phenotype of LEA1-disrupted yeast strains. (A) Growth behavior of LEA1- (Δlea1), YIB9- (Δyib9) or YIB9- and LEA1- (Δlea1 Δyib9) disrupted yeast strains compared with a wild-type control strain (WT) at 37°C. The arrow indicates the transfer from 30 to 37°C. (B) U2 snRNA levels in LEA1-disrupted yeast strains. U2 and U4 snRNA from extracts derived from Δlea1, Δyib9 and Δlea1 Δyib9 strains were analysed by primer extension. Download figure Download PowerPoint Lea1p is required for normal accumulation of the U2 snRNA As Lea1p is associated with the U2 snRNA (see above), we next analyzed the effect of LEA1 and/or the YIB9 disruptions on the level of U2 snRNA. Total RNA was extracted from the various disrupted strains grown at 30°C and analyzed by primer extension. This revealed reduced levels of U2 snRNA in the mutant strains compared with an isogenic wild-type control (Figure 3B). In contrast, the levels of U4 snRNA (Figure 3B) and of the other spliceosomal snRNAs (data not shown) were not affected by the disruption of the LEA1 and/or YIB9 gene. The three mutant strains had a 2.5-fold lower level of U2 snRNA compared with the isogenic wild-type strain. A comparable reduction of U2 snRNA has been reported previously for YIB9 (Tang et al., 1996). These data demonstrate that Lea1p and Yib9p are specifically required for U2 snRNA accumulation, as expected for snRNP proteins. Lea1p and Yib9p have identical effects on U2 snRNA level when disrupted alone or in combination, suggesting again that the two proteins have no independent function. Lea1p association with U2 snRNA is Yib9p-dependent The results presented above indicate that Lea1p is a component of the U2 snRNP. In the mammalian system, the simultaneous presence of U2B″ and U2A′ (the homologues of Yib9p and Lea1p, respectively) is required for interaction with U2 snRNA in vitro. In yeast, however, Yib9p was shown to interact with U2 snRNA on its own in vitro (Polycarpou-Schwarz et al., 1996; Tang et al., 1996). We therefore decided to test the requirement for Yib9p and Lea1p interaction with U2 snRNA in vivo. To examine whether Lea1p can interact with U2 snRNA in the absence of Yib9p, the YIB9 gene was disrupted in the strain harboring the Lea1–ProtA fusion (Δyib9 Lea1–ProtA). Conversely, to test whether Yib9p can interact with U2 snRNA in the absence of Lea1p, a plasmid carrying a ProtA–Yib9 fusion (Polycarpou-Schwarz et al., 1996) was introduced in the strain carrying the LEA1 gene disruption. Extracts were prepared from these two strains. Strains expressing the same tagged proteins in a non-mutated background and an isogenic wild-type were used as controls. Aliquots of these extracts were mixed with a similar volume of an extract containing the ProtA–Pop1 fusion, and the mixtures were incubated with IgG–agarose beads. Pellets of material bound to the beads were recovered and analyzed for their protein and RNA contents. The level of ProtA–Pop1 detected by Western blotting was similar in all lanes, indicating that all pellets had been efficiently recovered. Similar amounts of Lea1–ProtA were detected in the immunoprecipitates from the Lea1–ProtA and Δyib9 Lea1–ProtA extracts (Figure 4A, lanes 2 and 4). This result indicates that Lea1p is stable in the absence of Yib9p. Likewise, the similar levels of ProtA–Yib9 in pellets derived from extracts carrying or lacking Lea1p indicate that Yib9p was stable in the absence of Lea1p. A comparison of the levels of proteins present in input and pellet fractions also indicated that all proteins were efficiently precipitated (data not shown). Primer extension analysis revealed that U2 snRNA was efficiently precipitated by the tagged Lea1p only in the presence of Yib9p (Figure 4B, compare lanes 2 and 4). Surprisingly, Yib9p could also only coprecipitate U2 snRNA in the presence of Lea1p (Figure 4B, compare lanes 3 and 5). The lack of U2 snRNA signal in immunoprecipitates from the cells carrying the LEA1 or YIB9 disruption cannot be explained by the loss of the corresponding RNA pellets. Indeed, similar levels of the MRP RNA that associate with the Pop1–ProtA (Lygerou et al., 1994) were recovered in all samples. We therefore conclude that Lea1p and Yib9p need to be present simultaneously to allow stable interaction with U2 snRNA in vivo. Figure 4.Lea1p interacts with Yib9p and this interaction is required for association with U2 snRNA. (A) Extracts from a wild-type strain (WT) (lane 1), a strain with the integrated LEA1–ProtA construct (lane 2), a wild-type strain with the plasmid coding for the YIB9–ProtA fusion (lane 4), the YIB9-disrupted strain with the integrated LEA1–ProtA construct (lane 3) (Δyib9 LEA1–ProtA) or the LEA1-disrupted strain transformed with the plasmid harboring the YIB9–ProtA fusion (Δlea1 ProtA–YIB9) (lane 5) mixed with an extract harboring ProtA–Pop1 (all lanes), were immunoprecipitated with IgG–agarose beads. Proteins present in the pellet were analyzed by Western blotting. The signals corresponding to the different ProtA-tagged proteins are shown on the left while a molecular size marker is depicted on the right. (B) RNA was extracted from pellets described in (A) (lanes 1–5) and the presence of U2 snRNA and RNase MRP RNA (labeled MRP RNA; loading control) was assayed by primer extension. The lane numbers correspond to those in (A). (C) Interaction of Lea1p and Yib9p in vitro. Coprecipitation experiments were performed with Lea1p and Yib9p fused to either a His or GST using glutathione–agarose beads. GST alone and His-SmX6 served as negative controls. In lanes 1–6 the different proteins used were loaded according to their molecular size. Lanes 8–14 show the different combinations tested. The proteins were visualized by Coomassie staining. Download figure Download PowerPoint Lea1p interacts specifically and directly with Yib9p The data presented above suggest a significant similarity between the yeast Lea1p and Yib9p proteins and human U2A′ and U2B″. These latter two proteins have been shown to interact directly. To test whether this is also the case for Yib9p and Lea1p, we inserted the corresponding coding sequences in vectors suitable to test for protein–protein interaction in the yeast two-hybrid assay (Fields and Song, 1989). High levels of β-galactosidase activity indicated that Lea1p and Yib9p are interacting in this assay (Table I). This interaction was specific, as no homotypic binding of Lea1p or Yib9p or interaction with other snRNP proteins used as controls (SmE and SmG) could be detected (Table I). To confirm this result and show direct physical interactions between Lea1p and Yib9p, we conducted an in vitro binding study. We independently expressed recombinant Lea1p and Yib9p proteins fused to either a hexa-histidine (His) or glutathione S-transferase tag (GST). Aliquots of E.coli lysates containing the GST–Lea1p and GST–Yib9p, as well as a control lysate harboring only GST, were mixed with purified His-Lea1p, His-Yib9p or His-SmX6 proteins. After incubation with glutathione–agarose beads, the proteins specifically bound to the support were released, fractionated on a denaturing gel and detected by Coomassie staining (Figure 4C). The His-Lea1p protein was specifically coprecipitated by the GST–Yib9p fusion (Figure 4C, lane 11) but not by GST alone or GST–Lea1p (Figure 4C, lanes 8 and 14). Conversely, the His-Yib9p fusion coprecipitated with GST–Lea1p (Figure 4C, lane 13) but not with GST or GST–Yib9p (Figure 4C, lanes 7 and 10). Further evidence for the specificity of these coprecipitations comes from the analysis of the His-SmX6 protein that was not recovered with either of the two GST fusion proteins (Figure 4C, lanes 9 and 12). These results demonstrate that Lea1p and Yib9p are interacting directly and specifically with each other in the absence of other factors including U2 snRNA. Table 1. Yeast two-hybrid assay for Lea1p and Yib9p protein interaction pAS2ΔΔa Lea1pa Yib9pa SmEa pACTIIb 0.025 ± 0.004 0.018 ± 0.004 0.012 ± 0.002 0.032 ± 0.015 Lea1pb 0.023 ± 0.001 0.026 ± 0.006 55.8 ± 48.3 0.11 ± 0.007 Yib9pb 0.030 ± 0.004 12.7 ± 4.4 0.030 ± 0.005 0.027 ± 0.008 SmGb 0.027 ± 0.001 0.024 ± 0.001 0.020 ± 0.003 10.0 ± 9.1 a Constructs with a Gal4p DNA-binding domain (pAS2-derivatives). b Constructs with a transactivation domain (pACTII-derivatives). The standard error was calculated from two independent experiments. Significant activities are shown in bold. LEA1 disruption affects splicing in vivo Next, we analyzed whether Lea1p or Yib9p affected splicing in vivo, because the role of their human counterparts, U2A′ and U2B″, in splicing is unknown. For this purpose, strains deleted for the LEA1 gene, the YIB9 gene or an isogenic wild-type control were transformed with a reporter plasmid containing the RP51A intron inserted into the lacZ coding sequence or an empty control vector (Teem and Rosbash, 1983). Transformants were grown at 30°C, the reporter gene was induced and total RNA was extracted. The levels of pre-mRNA, mRNA and lariat intermediate were determined by primer extension (Figure 5). The wild-type strain contained high levels of mRNA and low levels of pre-mRNA (Figure 5, lane 4). In contrast, cells lacking Lea1p or Yib9p harbored reduced levels of mRNA and accumulated pre-mRNA (Figure 5, lanes 5 and 6). Assaying for the β-galactosidase produced by these reporters confirmed this result (data not shown). Quantification of the extension products revealed that the ratio of mRNA to pre-mRNA (a measurement of splicing efficiency; Pikielny and Rosbash, 1985) was reduced ∼20-fold in the mutant strains (M/P; Figure 5, bottom). The ratio of lariat intermediate to pre-mRNA was reduced by the same proportion while the ratio of mRNA to lariat intermediate was similar for the three strains, indicating that the first splicing step was specifically affected (Fouser and Friesen, 1986). We conclude that Lea1p and Yib9p are required for an efficient first step of splicing in vivo. Figure 5.LEA1 disruption affects splicing in vivo. A reporter construct (reporter) or a plasmid lacking the reporter construct (vector) were introduced into a wild-type (WT), a Δlea1 or a Δyib9 strain. RNA was extracted and splicing was analyzed by primer extension using the exon 2 specific EM38 primer. The reporter construct contains the wild-type RP51A intron inserted into the β-galactosidase coding sequence. The different extension products are labeled on the right of the figure. Signals were quantified using a PhosphorImager. The ratio between mRNA and pre-mRNA was calculated and is indicated below the figure (M/P). Signals in control lanes 1–3 were not quantified. Download figure Download PowerPoint Lea1p is required for spliceosome assembly To define the nature of the splicing block conferred by the absence of Lea1p (and Yib9p), we analyzed spliceosome assembly in vitro. Splicing extracts prepared from wild-type, Δlea1, Δyib9 and Δlea1 Δyib9 strains were incubated with a radioactively labeled pre-mRNA in either the presence or absence of ATP, and splicing complex formation was assayed by native gel electrophoresis (Figure 6). In the absence of ATP, CC2 accumulated in a wild-type extract (Figure 6, lane 1) as reported previously (Séraphin and Rosbash, 1989). In these conditions, CC2 also formed efficiently in extracts prepared from the mutant strains, indicating that the absence of Lea1p or Yib9p did not affect early splicing complex formation (Figure 6, lane 2–4). This is consistent with the observation that U2 snRNP is not required for commitment complex assembly (Séraphin and Rosbash, 1989). In the presence of ATP, spliceosome assembled efficiently in the extract prepared from a wild-type strain (Figure 6, lane 5). Interestingly, extracts prepared from the mutant strains were consistently unable to form spliceosome under these conditions (Figure 6, lanes 6–8) and accumulated CC2. A similar observation was made independently following the disruption of YIB9 (Tang et al., 1996). The absence of spliceosome in the mutant extracts suggested that Lea1p and Yib9p affect addition of U2 snRNP to CC2. An early block in spliceosome assembly is consistent with our in vivo observation that Lea1p is required for the first splicing step. However, the in vitro effect could be indi" @default.
• W2112654577 created "2016-06-24" @default.
• W2112654577 creator A5023117880 @default.
• W2112654577 creator A5083517070 @default.
• W2112654577 date "1998-11-02" @default.
• W2112654577 modified "2023-09-30" @default.
• W2112654577 title "The yeast U2A′/U2B″ complex is required for pre-spliceosome formation" @default.
• W2112654577 cites W1508168356 @default.
• W2112654577 cites W1553769217 @default.
• W2112654577 cites W1597533137 @default.
• W2112654577 cites W1849895108 @default.
• W2112654577 cites W1905489680 @default.
• W2112654577 cites W1907972331 @default.
• W2112654577 cites W1941660249 @default.
• W2112654577 cites W1964571492 @default.
• W2112654577 cites W1967554461 @default.
• W2112654577 cites W1967944402 @default.
• W2112654577 cites W1968276418 @default.
• W2112654577 cites W1977186059 @default.
• W2112654577 cites W1986204780 @default.
• W2112654577 cites W1988498926 @default.
• W2112654577 cites W1989331281 @default.
• W2112654577 cites W1992097120 @default.
• W2112654577 cites W1993926172 @default.
• W2112654577 cites W2002962124 @default.
• W2112654577 cites W2003213264 @default.
• W2112654577 cites W2003277334 @default.
• W2112654577 cites W2005123682 @default.
• W2112654577 cites W2006062026 @default.
• W2112654577 cites W2008621771 @default.
• W2112654577 cites W2009310436 @default.
• W2112654577 cites W2010696389 @default.
• W2112654577 cites W2022176376 @default.
• W2112654577 cites W2024267851 @default.
• W2112654577 cites W2026137624 @default.
• W2112654577 cites W2033883110 @default.
• W2112654577 cites W2036231394 @default.
• W2112654577 cites W2052319720 @default.
• W2112654577 cites W2054306198 @default.
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• W2112654577 cites W2056200374 @default.
• W2112654577 cites W2064448615 @default.
• W2112654577 cites W2080408951 @default.
• W2112654577 cites W2082468024 @default.
• W2112654577 cites W2084597963 @default.
• W2112654577 cites W2085005364 @default.
• W2112654577 cites W2087137984 @default.
• W2112654577 cites W2097382368 @default.
• W2112654577 cites W2098189749 @default.
• W2112654577 cites W2099158977 @default.
• W2112654577 cites W2124049693 @default.
• W2112654577 cites W2124605498 @default.
• W2112654577 cites W2128521583 @default.
• W2112654577 cites W2132505931 @default.
• W2112654577 cites W2142957866 @default.
• W2112654577 cites W2155322043 @default.
• W2112654577 cites W2163793704 @default.
• W2112654577 cites W2165543326 @default.
• W2112654577 cites W2166992176 @default.
• W2112654577 cites W2167557755 @default.
• W2112654577 cites W2229897834 @default.
• W2112654577 cites W2272248373 @default.
• W2112654577 cites W2294055356 @default.
• W2112654577 cites W323731211 @default.
• W2112654577 cites W41372527 @default.
• W2112654577 cites W4232816298 @default.
• W2112654577 cites W92004496 @default.
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