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.     

The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex.

The yeast PRP44 gene, alternatively named as BRR2, SLT22, RSS1, or SNU246, encodes a 246-kDa protein with putative RNA helicase function during pre-mRNA splicing. The protein is a typical DEAD/H family member, but unlike most other members of this family, it contains two putative RNA helicase domains, each with a highly conserved ATPase motif. Prior to this study little was known about functional roles for these two domains. We present genetic and biochemical evidence that ATPase motifs of only the first helicase domain are required for cell viability and pre-mRNA splicing. Overexpression of mutations in the first domain results in a dominant negative phenotype, and extracts from these mutant strains inhibit in vitro pre-mRNA splicing. In vitro analyses of affinity purified proteins revealed that only the first helicase domain possesses poly (U)-dependent ATPase activity. Overexpression of a dominant negative protein in vivo reduces the relative abundance of free U4 and U6 snRNA with a concomitant accumulation of the U4/U6 duplex. Accumulation of the U4/U6 duplex was relieved by overexpression of wild-type Prp44p. Three DEAD/H box proteins, Prp16p, Prp22p and Prp44p, have previously been shown to affect U4/U6 unwinding activity in vitro. The possible role of these proteins in mediating this reaction in vivo was explored following induced expression of ATPase domain mutants in each of these. Although overexpression of the mutant form of either Prp16p, Prp22p, or Prp44p was lethal, only expression of the mutant Prp44p resulted in accumulation of the U4/U6 helix. Our results, when combined with previously published in vitro results, support a direct role for Prp44p in unwinding of the U4/U6 helix.     

The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes.     

Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome

The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.     

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.     

Characterization of U6 snRNA-protein interactions

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.     

p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4-U6 and U6-U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110(nrb) or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri-snRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.     

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.     

Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs.

Prp24 was previously isolated as a suppressor of a cold-sensitive U4 mutation and is required for at least the first step of splicing in vitro. Our investigation of the in vitro RNA binding properties of the purified Prp24 protein shows that it binds preferentially to the U4/U6 hybrid snRNAs compared to other snRNAs. The interaction between Prp24 and the U4/U6 hybrid appears to involve two regions in the RNA: the 39-57 region of U6 and stem II of the U4/U6 hybrid. Interestingly, some U4 mutations, which destabilize stem II, increase the affinity of Prp24 for the U4/U6 RNAs compared to the wild type. This suggests that the binding of Prp24 to the U4/U6 RNAs may involve some destabilization of the RNA duplex. We also found that Prp24 can stimulate the annealing of U4 and U6, suggesting that Prp24 participates in both the formation and disassembly of the U4/U6 hybrid during splicing.     

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.     

A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs.

We have purified three new human U4/U6-snRNP proteins from HeLa cells. The three proteins formed a tightly bound complex and behaved as a single species throughout the purification. All three proteins have been identified by peptide sequencing, and full-length cDNA sequences have been obtained for all of them. Two of the proteins are homologues of the Saccharomyces cerevisiae splicing factors Prp3 and Prp4, and the third protein is a cyclophilin. Both the human and S. cerevisiae Prp4 proteins have seven repeats of the WD motif and likely fold into structures very similar to those of the beta subunits of G proteins. The human Prp3 protein is highly basic and is closely related to S. cerevisiae Prp3 only in its carboxyl-terminal half. The human homologues of Prp3 and Prp4 are part of a stable complex in the absence of RNA. The third protein in the complex is a new cyclophilin. Cyclophilins have been proposed to act as chaperones in a variety of cellular processes, and we discuss some possible roles of this U4/U6 snRNP-associated cyclophilin.     

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.     

Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements.

U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end. The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing. Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24. Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix. These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle.     

LSM1 over-expression in Saccharomyces cerevisiae depletes U6 snRNA levels

Lsm1 is a component of the Lsm1-7 complex involved in cytoplasmic mRNA degradation. Lsm1 is over-expressed in multiple tumor types, including over 80% of pancreatic tumors, and increased levels of Lsm1 protein have been shown to induce carcinogenic effects. Therefore, understanding the perturbations in cell process due to increased Lsm1 protein may help to identify possible therapeutics targeting tumors over-expressing Lsm1. Herein, we show that LSM1 over-expression in the yeast Saccharomyces cerevisiae inhibits growth primarily due to U6 snRNA depletion, thereby altering pre-mRNA splicing. The decrease in U6 snRNA levels causes yeast strains over-expressing Lsm1 to be hypersensitive to loss of other proteins required for production or function of the U6 snRNA, supporting a model wherein excess Lsm1 reduces the availability of the Lsm2-7 proteins, which also assemble with Lsm8 to form a complex that binds and stabilizes the U6 snRNA. Yeast strains over-expressing Lsm1 also display minor alterations in mRNA decay and demonstrate increased susceptibility to mutations inhibiting cytoplasmic deadenylation, a process required for both 5′-to-3′ and 3′-to-5′ pathways of exonucleolytic decay. These results suggest that inhibition of splicing and/or deadenylation may be effective therapies for Lsm1-over-expressing tumors.     

The Prp19-associated complex is required for specifying interactions of U5 and U6 with pre-mRNA during spliceosome activation

Activation of the spliceosome involves a major structural change in the spliceosome, including release of U1 and U4 small nuclear ribonucleoprotein particles and the addition of a large protein complex, the Prp19-associated complex. We previously showed that the Prp19-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is released. Changes within the spliceosome upon binding of the Prp19-associated complex include remodeling of the U6/5' splice site interaction and destabilization of Lsm proteins to allow further interaction of U6 with the intron sequence. Here, we further analyzed interactions of U5 and U6 with pre-mRNA at various stages of spliceosome assembly from initial binding of tri-small nuclear ribonucleoprotein complex to the activated spliceosome to reveal stepwise changes of interactions. We demonstrate that both U5 and U6 interacted with pre-mRNA in dynamic manners spanning over a large region of U6 and the 5' exon sequences prior to the activation of the spliceosome. During spliceosome activation, interactions were locked down to small regions, and the Prp19-associated complex was required for defining the specificity of interaction of U5 and U6 with the 5' splice site to stabilize their association with the spliceosome after U4 is dissociated.     

Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation

The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.     

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.     

Characterization of Sm-like proteins in yeast and their association with U6 snRNA.

Seven Sm proteins associate with U1, U2, U4 and U5 spliceosomal snRNAs and influence snRNP biogenesis. Here we describe a novel set of Sm-like (Lsm) proteins in Saccharomyces cerevisiae that interact with each other and with U6 snRNA. Seven Lsm proteins co-immunoprecipitate with the previously characterized Lsm4p (Uss1p) and interact with each other in two-hybrid analyses. Free U6 and U4/U6 duplexed RNAs co-immunoprecipitate with seven of the Lsm proteins that are essential for the stable accumulation of U6 snRNA. Analyses of U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs in Lsm-depleted strains suggest that Lsm proteins may play a role in facilitating conformational rearrangements of the U6 snRNP in the association-dissociation cycle of spliceosome complexes. Thus, Lsm proteins form a complex that differs from the canonical Sm complex in its RNA association(s) and function. We discuss the possible existence and functions of alternative Lsm complexes, including the likelihood that they are involved in processes other than pre-mRNA splicing.     

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.