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.     

A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles

The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure. The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small nuclear RNAs (snRNAs). Here, the yeast RNA binding protein Prp24 is shown to reanneal these snRNAs. When Prp24 is absent, unpaired U4 and U6 small nuclear ribonucleoprotein particles (snRNPs) accumulate; with time, splicing becomes inhibited. Addition of purified Prp24 protein regenerates duplex U4/U6 snRNPs for new rounds of splicing. The reannealing reaction catalyzed by Prp24 proceeds more efficiently with snRNPs than with deproteinized snRNAs.     

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.     

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.     

Modulation of E-cadherin expression in TPA-induced cell motility: well-differentiated human adenocarcinoma cells move as coherent sheets associated with phosphorylation of E-cadherin-catenin complex

We have identified a human splicing factor required for the second step of pre-mRNA splicing. This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18. In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished. Splicing activity is restored by the addition of recombinant hPrp18, demonstrating that hPrp18 is required for the second step. The hPrp18 protein is bound tightly to the spliceosome only during the second step of splicing. hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans. Splicing activity can be restored to hPrp18-depleted HeLa cell extracts by yeast Prp18, showing that important functional regions of the proteins have been conserved. A 90-amino-acid region near the carboxyl terminus of hPrp18 is strongly homologous to yeast Prp18 and is also conserved in rice and nematodes. The homology identifies one region important for the function of both proteins and may define a new protein motif. In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs. A 55-kD protein that cross-reacts with antibodies against hPrp18 is a constituent of the U4/U6 and U4/U6 x U5 snRNP particles.     

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.     

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.     

RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2

BACKGROUND: The dynamic rearrangements of RNA structure which occur during pre-mRNA splicing are thought to be mediated by members of the DExD/H-box family of RNA-dependent ATPases. Although three DExD/H-box splicing factors have recently been shown to unwind synthetic RNA duplexes in purified systems, in no case has the natural biological substrate been identified. A duplex RNA target of particular interest is the extensive base-pairing interaction between U4 and U6 small nuclear RNAs. Because these helices must be disrupted to activate the spliceosome for catalysis, this rearrangement is believed to be tightly regulated in vivo. RESULTS: We have immunopurified Brr2, a DEIH-box ATPase, in a native complex containing U1, U2, U5 and duplex U4/U6 small nuclear ribonucleoprotein particles (snRNPs). Addition of hydrolyzable ATP to this complex results in the disruption of U4/U6 base-pairing, and the release of free U4 and U6 snRNPs. A mutation in the helicase-like domain of Brr2 (brr2-1) prevents these RNA rearrangements. Notably, U4/U6 dissociation and release occur in the absence of exogenously added pre-mRNA. CONCLUSIONS: Disruption of U4/U6 base-pairing in native snRNPs requires ATP hydrolysis and Brr2. This is the first assignment of a DExD/H-box splicing factor to a specific biological unwinding event. The unwinding function of Brr2 can be antagonized by the annealing activity of Prp24. We propose the existence of a dynamic cycle, uncoupled from splicing, that interconverts free and base-paired U4/U6 snRNPs.     

Dissection of the DNA binding domain of yeast Zn-finger protein Rme1p, a repressor of meiotic activator IME1

A series of deletion mutants of the yeast Zn-finger protein Rme1p (Repressor of Meiosis) fused with maltose binding protein (MBP) were constructed, purified, and characterized to examine the DNA binding domain. It was shown by gel retardation assay that the DNA binding domain of Rme1p was attributed to C-terminal amino acid residues 171 to 300. All three Zn-fingers are involved in the DNA binding domain, but they are not sufficient for DNA binding ability. Notably, the C-terminal region (residues 285-300) is essential for DNA binding. Provided that the region folds into alpha-helix, the basic amino acid residues may form a ridge on one side of the helix, whereas the hydrophobic residues may form it on the other side. Thus, the DNA binding domain of Rme1p would be dissected two regions. The roles of C-terminal region in DNA recognition will be discussed.     

Essential domains of the PRP21 splicing factor are implicated in the binding to PRP9 and PRP11 proteins and are conserved through evolution

The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes. This factor is required for the assembly of the prespliceosome. Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown. Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module. Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors. In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants. Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p. Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant. Deletion analyses led to the definition of domains required for viability. These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution. They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.     

U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.     

Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence

The translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N- and C-terminal splice junctions and around the N-terminal one-third of VDE. We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His362, newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His736 assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE.     

Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8

During the splicing process, spliceosomal snRNAs undergo numerous conformational rearrangements that appear to be catalyzed by proteins belonging to the DEAD/H-box superfamily of RNA helicases. We have cloned a new RNA helicase gene, designated DBP2 (DEAH-boxprotein), homologous to the Schizosaccaromyces pombe cdc28(+)/prp8(+) gene involved in pre-mRNA splicing and cell cycle progression. The full-length DBP2 contains 3400 nucleotides and codes for a protein of 1041 amino acids with a calculated mol. wt of 119 037 Da. Transfection experiments demonstrated that the GFP-DBP2 gene product, transiently expressed in HeLa cells, was localized in the nucleus. The DBP2 gene was mapped by FISH to the MHC region on human chromosome 6p21.3, a region where many malignant, genetic and autoimmune disease genes are linked. Because the expression of DBP2 gene in S.pombe prp8 mutant cells partially rescued the temperature-sensitive phenotype, we conclude that DBP2 is a functional human homolog of the fission yeast Cdc28/Prp8 protein.     

Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing

Pre-mRNA splicing takes place in two catalytic steps. The second step is poorly understood, especially in mammals. In yeast, the splicing factors, Prps 16, 17, 18 and Slu7 function exclusively in step II. Here we report the isolation of cDNAs encoding human Prps 16 and 17 which are 41 and 36% identical to their yeast counterparts. The Prp16 gene is essential in yeast, and we show that a chimeric yeast-human Prp16 protein rescues a yeast Prp16 knockout strain. Immunodepletion of hPrp16 from splicing extracts specifically blocks step II, and the activity can be fully restored with recombinant hPrp16. Moreover, both hPrps 16 and 17 associate with the spliceosome late in the splicing pathway. Mutations at the 3' splice site that specifically block step II do not affect the association of hPrps 16 and 17 with the spliceosome, indicating that these factors may function at a stage of step II prior to recognition of the 3' splice site. Recently, the human homologs of Prp18 and Slu7 were identified. The observation that humans contain homologs of all four known step II proteins in yeast indicates that the mechanism for catalytic step II is highly conserved.     

The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression

The dbf3 mutation was originally obtained in a screen for DNA synthesis mutants with a cell cycle phenotype in the budding yeast Saccharomyces cerevisiae. We have now isolated the DBF3 gene and found it to be an essential gene with an ORF of 7239 nucleotides, potentially encoding a large protein of 268 kDa. We also obtained an allele-specific high copy number suppressor of the dbf3-1 allele, encoded by the known SSB1 gene, a member of the Hsp70 family of heat shock proteins. The sequence of the Dbf3 protein is 58% identical over 2300 amino acid residues to a predicted protein from Caenorhabditis elegans. Furthermore, partial sequences with 61% amino acid sequence identity were deduced from two files of human cDNA in the EST nucleotide database so that Dbf3 is a highly conserved protein. The nucleotide sequence of DBF3 turned out to be identical to the yeast gene PRP8, which encodes a U5 snRNP required for pre-mRNA splicing. This surprising result led us to further characterise the phenotype of dbf3 which confirmed its role in the cell cycle and showed it to function early, around the time of S phase. This data suggests a hitherto unexpected link between pre-mRNA splicing and the cell cycle.     

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.     

Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein

A 429 aa theoretical intein is encoded in the dnaB gene (DNA helicase) of the cyanobacterium Synechocystis sp. strain PCC6803. This intein is shown to be capable of protein splicing with or without its native exteins when tested in E. coli cells. A centrally located 275 amino acid sequence (residues 107-381) of this intein can be deleted without loss of the protein splicing activity, resulting in a functional mini-intein of 154 aa in size. Efficient in vivo protein trans-splicing was observed when this mini-intein was split into a 106 aa N-terminal fragment containing intein motifs A and B, and a 48 aa C-terminal fragment containing intein motifs F and G. These results indicate that the N- and C-terminal regions of the Ssp DnaB intein, whether covalently linked with each other or not, can come together through non-covalent interaction to form a protein splicing domain that is functionally sufficient and structurally independent from the centrally located endonuclease domain of the intein.