Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

Assembly of Snu114 into U5 snRNP requires Prp8 and a functional GTPase domain

Snu114 is a U5 snRNP protein essential for pre-mRNA splicing. Based on its homology with the ribosomal translocase EF-G, it is thought that GTP hydrolysis by Snu114 induces conformational rearrangements in the spliceosome. We recently identified allele-specific genetic interactions between SNU114 and genes encoding three other U5 snRNP components, Prp8 and two RNA-dependent ATPases, Prp28 and Brr2, required for destabilization of U1 and U4 snRNPs prior to catalysis. To shed more light onto the function of Snu114, we have now directly analyzed snRNP and spliceosome assembly in SNU114 mutant extracts. The Snu114-60 C-terminal truncation mutant, which is synthetically lethal with the ATPase mutants prp28-1 and brr2-1, assembles spliceosomes but subsequently blocks U4 snRNP release. Conversely, mutants in the GTPase domain fail to assemble U5 snRNPs. These mutations prevent the interaction of Snu114 with Prp8 as well as with U5 snRNA. Since Prp8 is thought to regulate the activity of the DEAD-box ATPases, this strategy of snRNP assembly could ensure that Prp8 activity is itself regulated by a GTP-dependent mechanism.     

The U5 snRNA internal loop 1 is a platform for Brr2, Snu114 and Prp8 protein binding during U5 snRNP assembly

The U5 small nuclear ribonucleoprotein particle (snRNP) forms the heart of the spliceosome which is required for intron removal from pre‐mRNA. The proteins Prp8, Snu114 and Brr2 all assemble with the U5 small nuclear RNA (snRNA) to produce the U5 snRNP. Successful assembly of the U5 snRNP, then incorporation of this snRNP into the U4/U6.U5 tri‐snRNP and the spliceosome, is essential for producing an active spliceosome. We have investigated the requirements for Prp8, Snu114 and Brr2 association with the U5 snRNA to form the U5 snRNP in yeast. Mutations were constructed in the highly conserved loop 1 and internal loop 1 (IL1) of the U5 snRNA and their function assessed in vivo. The influence of these U5 mutations on association of Prp8, Snu114 and Brr2 with the U5 snRNA were then determined. U5 snRNA loop 1 and both sides of IL1 in U5 were important for association of Prp8, Snu114 and Brr2 with the U5 snRNA. Mutations in the 3′ side of U5 IL1 resulted in the greatest reduction of Prp8, Snu114 and Brr2 association with the U5 snRNA. Genetic screening of brr2 and U5 snRNA mutants revealed synthetic lethal interactions between alleles in Brr2 and the 3′ side of U5 snRNA IL1 which reflects reduced association between Brr2 and U5 IL1. We propose that the U5 snRNA IL1 is a platform for protein binding and is required for Prp8, Brr2 and Snu114 association with the U5 snRNA to form the U5 snRNP. J. Cell. Biochem. 114: 2770–2784, 2013. © 2013 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Mer1p is a modular splicing factor whose function depends on the conserved U2 snRNP protein Snu17p

Mer1p activates the splicing of at least three pre-mRNAs (AMA1, MER2, MER3) during meiosis in the yeast Saccharomyces cerevisiae. We demonstrate that enhancer recognition by Mer1p is separable from Mer1p splicing activation. The C-terminal KH-type RNA-binding domain of Mer1p recognizes introns that contain the Mer1p splicing enhancer, while the N-terminal domain interacts with the spliceosome and activates splicing. Prior studies have implicated the U1 snRNP and recognition of the 5′ splice site as key elements in Mer1p-activated splicing. We provide new evidence that Mer1p may also function at later steps of spliceosome assembly. First, Mer1p can activate splicing of introns that have mutated branch point sequences. Secondly, Mer1p fails to activate splicing in the absence of the non-essential U2 snRNP protein Snu17p. Thirdly, Mer1p interacts with the branch point binding proteins Mud2p and Bbp1p and the U2 snRNP protein Prp11p by two-hybrid assays. We conclude that Mer1p is a modular splicing regulator that can activate splicing at several early steps of spliceosome assembly and depends on the activities of both U1 and U2 snRNP proteins to activate splicing.     

Physical and genetic interactions of yeast Cwc21p,an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors—namely, Prp8p and Snu114p—and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.     

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 comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.     

A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly

We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.     

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.     

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.     

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.     

Prp8p dissection reveals domain structure and protein interaction sites

We describe a novel approach to characterize the functional domains of a protein in vivo. This involves the use of a custom-built Tn5-based transposon that causes the expression of a target gene as two contiguous polypeptides. When used as a genetic screen to dissect the budding yeast PRP8 gene, this showed that Prp8 protein could be dissected into three distinct pairs of functional polypeptides. Thus, four functional domains can be defined in the 2413-residue Prp8 protein, with boundaries in the regions of amino acids 394-443, 770, and 2170-2179. The central region of the protein was resistant to dissection by this approach, suggesting that it represents one large functional unit. The dissected constructs allowed investigation of factors that associate strongly with the N- or the C-terminal Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437-770, whereas fragmenting Prp8p at residue 2173 destabilizes its association with Aar2p.     

Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus

Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking. Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.     

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 splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.     

The Clf1p splicing factor promotes spliceosome assembly through N-terminal tetratricopeptide repeat contacts

Spliceosome assembly follows a well conserved pathway of subunit addition that includes both small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splicing factors. Clf1p is an unusual splicing factor composed almost entirely of direct repeats of the tetratricopeptide repeat (TPR) protein-binding motif. Here we show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex (nineteen complex; NTC) and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs. Thirty Ccf (Clf1p complex factor) proteins have been identified by mass spectroscopy or immune detection as known or suspected components of the yeast spliceosome. Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p protein (i.e. Clf1Delta2-TAP) destabilizes Clf1p complexes assembled in vivo, causing the release of the Cef1p and Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and Hsh155p snRNP proteins. In vitro, temperature inactivation of Clf1Delta2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex. These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.     

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.     

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.