The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP.

Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing. No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts. Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP. Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs. Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions. Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly. These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs. Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs. Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans.     

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.     

3'-cyclic phosphorylation of U6 snRNA leads to recruitment of recycling factor p110 through LSm proteins

Pre-mRNA splicing proceeds through assembly of the spliceosome complex, catalysis, and recycling. During each cycle the U4/U6.U5 tri-snRNP is disrupted and U4/U6 snRNA base-pairing unwound, releasing separate post-spliceosomal U4, U5, and U6 snRNPs, which have to be recycled to the splicing-competent tri-snRNP. Previous work implicated p110--the human ortholog of the yeast Prp24 protein--and the LSm2-8 proteins of the U6 snRNP in U4/U6 recycling. Here we show in vitro that these proteins bind synergistically to U6 snRNA: Both purified and recombinant LSm2-8 proteins are able to recruit p110 protein to U6 snRNA via interaction with the highly conserved C-terminal region of p110. Furthermore, the presence of a 2',3'-cyclic phosphate enhances the affinity of U6 snRNA for the LSm2-8 proteins and inversely reduces La protein binding, suggesting a direct role of the 3'-terminal phosphorylation in RNP remodeling during U6 biogenesis.     

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6.U5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2.U4/U6.U5] tetra-snRNP complex.     

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.     

Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae.

The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.     

Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6*U5 tri-snRNP formation and pre-mRNA splicing

In each round of nuclear pre-mRNA splicing, the U4/U6*U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6*U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing. Significantly, complementation with recombinant 61K protein restores each of these steps. Protein 61K is operationally defined as U4/U6 snRNP-specific as it remains bound to this particle at salt concentrations where the tri-snRNP dissociates. However, as shown by two-hybrid analysis and biochemical assays, protein 61K also interacts specifically with the U5 snRNP-associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the tri-snRNP. Interestingly, protein 61K is encoded by a gene (PRPF31) that has been shown to be linked to autosomal dominant retinitis pigmentosa. Thus, our studies suggest that disruptions in tri-snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease.     

The human homologue of the yeast splicing factor prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions

An essential step of pre-mRNA spliceosome assembly is the interaction between the snRNPs U4/U6 and U5, to form the [U4/U6.U5] tri-snRNP. While the tri-snRNP protein Prp6p appears to play an important role for tri-snRNP formation in yeast, little is known about the interactions that connect the two snRNP particles in human tri-snRNPs. Here, we describe the molecular characterisation of a 102kD protein form HeLa tri-snRNPs. The 102kD protein exhibits a significant degree of overall homology with the yeast Prp6p, including the conservation of multiple tetratrico peptide repeats (TPR), making this the likely functional homologue of Prp6p. However, while the yeast Prp6p is considered to be a U4/U6-specific protein, the human 102kD protein was found to be tightly associated with purified 20 S U5 snRNPs. This association appears to be primarily due to protein-protein interactions. Interestingly, antibodies directed against the C-terminal TPR elements of the 102kD protein specifically and exclusively immunoprecipitate free U5 snRNPs, but not [U4/U6.U5] tri-snRNPs, from HeLa nuclear extract, suggesting that the C-terminal region of the 102kD protein is covered by U4/U6 or tri-snRNP-specific proteins. Since proteins containing TPR elements are typically involved in multiple protein-protein interactions, we suggest that the 102kD protein interacts within the tri-snRNP with both the U5 and U4/U6 snRNPs, thus bridging the two particles. Consistent with this idea, we show that in vitro translated U5-102kD protein binds to purified 13S U4/U6 snRNPs, which contain, in addition to the Sm proteins, all known U4/U6-specific proteins.     

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p.

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.     

The 5' and 3' domains of yeast U6 snRNA: Lsm proteins facilitate binding of Prp24 protein to the U6 telestem region

The 5' and 3' domains of yeast U6 snRNA contain sequences that are thought to be important for binding to Prp24 and Lsm proteins. By extensive mutational analysis of yeast U6 snRNA, we confirmed that the 3' terminal uridine tract of U6 snRNA is important for U6 binding to Lsm proteins in yeast. Binding of Prp24 protein to U6 RNA is dependent on or is strongly enhanced by U6 binding of Lsm proteins. This supports a model for U6 snRNP assembly in which U6 RNA binds to the Lsm2-8 core prior to binding Prp24 protein. Using compensatory base-pairing analysis, we show that at least half of the recently identified U6 telestem as well as a nucleotide sequence in the other half of the telestem are important for binding of U6 RNA to Prp24 protein. Surprisingly, disruption of base pairing in the unconfirmed half of the telestem enhanced U6-Prp24 binding. Truncation of the entire 3' terminal domain or nearly the entire 5' terminal domain of yeast U6 allowed for detectable levels of splicing to proceed in vitro. In addition to gaining knowledge of the function of the 5' and 3' domains of yeast U6, our results help define the minimal set of requirements for yeast U6 RNA function in splicing. We present a revised secondary structural model of yeast U6 snRNA in free U6 snRNPs.     

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation.

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.     

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.     

The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure.     

Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.     

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

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.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.     

The human Prp8 protein is a component of both U2- and U12-dependent spliceosomes.

This study reports the cloning, sequencing, and development of antisera against the human U5 snRNP 220-kDa protein or hPrp8p. Prp8p is the most highly conserved large nuclear protein known to date, but it is not related to any other protein. Southern, Northern, and expressed sequence tag analyses indicate that hPrp8p is encoded by a single gene. Prp8p is a core component of U5 snRNP and the U4/U6.U5 tri-snRNP, and antibodies raised against it immunoprecipitate both the major, U2-dependent and minor, U12-dependent spliceosomes. These spliceosomes, which excise different classes of introns, contain distinct sets of snRNAs overlapping only with U5 snRNA. Other than the core Sm proteins, hPrp8p is the first splicing factor shown to be common to both spliceosomes.     

Mutations within the yeast U4/U6 snRNP protein Prp4 affect a late stage of spliceosome assembly.

We showed previously that the yeast Prp4 protein is a spliceosomal factor that is tightly associated with the U4, U5, and U6 small nuclear RNAs. Moreover, Prp4 appears to associate very transiently with the spliceosome before the U4 snRNA dissociates from the spliceosome. Prp4 belongs to the Gbeta-like protein family, which suggests that the Prp4 Gbeta motifs could mediate interactions with other components of the spliceosome. To investigate the function of the Gbeta motifs, we introduced mutations within the second WD-repeat of Prp4. Among the 35 new alleles found, 24 were pseudo wild-type mutants, 8 failed to grow at any temperature, and 3 were conditional sensitive mutants. The biochemical defects of the three thermosensitive prp4 mutants have been examined by immunoprecipitation, native gel electrophoresis, and glycerol gradient centrifugation. First, we show that snRNP formation is not impaired in these mutants and that Prp4 is present in the U4/U6 and U4/U6-U5 snRNP particles. We also demonstrate that spliceosome assembly is largely unaffected despite the fact that the first step of splicing does not occur. However, both Prp4 and U4 snRNA remain tightly associated with the spliceosome and this blocks the transition toward an active form of the spliceosome. Our results suggest a possible role of Prp4 in mediating important conformational rearrangements of proteins within the spliceosome that involve the region containing the Gbeta-repeats.     

A conditional U5 snRNA mutation affecting pre-mRNA splicing and nuclear pre-mRNA retention identifies SSD1/SRK1 as a general splicing mutant suppressor.

A combination of point mutations disrupting both stem 1 and stem 2 of U5 snRNA (U5AI) was found to confer a thermosensitive phenotype in vivo. In a strain expressing U5AI, pre-mRNA splicing was blocked before the first step through an inability of the mutant U5 snRNA to efficiently associate with the U4/U6 di-snRNP. Formation of early splicing complexes was not affected in extracts prepared from U5 snRNA mutant cells, while the capacity of these extracts to splice a pre-mRNA in vitro was greatly diminished. In addition, significant levels of a translation product derived from intron containing pre-mRNAs could be detected in vivo. The SSD1/SRK1 gene was identified as a multi-copy suppressor of the U5AI snRNA mutant. Single copy expression of SSD1/SRK1 was sufficient to suppress the thermosensitive phenotype, and high copy expression partially suppressed the splicing and U4/U6.U5 tri-snRNP assembly pheno-types. SSD1/SRK1 also suppressed thermosensitive mutations in the Prp18p and U1-70K proteins, while inhibiting growth of the cold sensitive U1-4U snRNA mutant at 30 degrees C. Thus we have identified SSD1/SRK1 as a general suppressor of splicing mutants.