The Prp1(+) Gene Required for Pre-mRNA Splicing in Schizosaccharomyces Pombe Encodes a Protein That Contains Tpr Motifs and Is Similar to Prp6p of Budding Yeast

The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)(+) RNA transport. The prp1(+) gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prp1p shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1(+) and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1(+) gene was found to be identical with the zer1(+) gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)(+) RNA nuclear export, in addition to pre-mRNA splicing.     

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.     

Functional domains of the yeast splicing factor Prp22p

The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase. Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome. Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities. Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro. The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p. The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage. We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome. We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth. However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.     

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.     

Isolation of an essential Schizosaccharomyces pombe gene, prp31(+), that links splicing and meiosis

We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31⁺ gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation.     

Extragenic Suppressors of Saccharomyces Cerevisiae Prp4 Mutations Identify a Negative Regulator of Prp Genes

The PRP4 gene encodes a protein that is a component of the U4/U6 small nuclear ribonucleoprotein particle and is necessary for both spliceosome assembly and pre-mRNA splicing. To identify genes whose products interact with the PRP4 gene or gene product, we isolated second-site suppressors of temperature-sensitive prp4 mutations. We limited ourselves to suppressors with a distinct phenotype, cold sensitivity, to facilitate analysis of mutants. Ten independent recessive suppressors were obtained that identified four complementation groups, spp41, spp42, spp43 and spp44 (suppressor of prp4, numbers 1-4). spp41-spp44 suppress the pre-mRNA splicing defect as well as the temperature-sensitive phenotype of prp4 strains. Each of these spp mutations also suppresses prp3; spp41 and spp42 suppress prp11 as well. Neither spp41 nor spp42 suppresses null alleles of prp3 or prp4, indicating that the suppression does not occur via a bypass mechanism. The spp41 and spp42 mutations are neither allele- nor gene-specific in their pattern of suppression and do not result in a defect in pre-mRNA splicing. Thus the SPP41 and SPP42 gene products are unlikely to participate directly in mRNA splicing or interact directly with Prp3p or Prp4p. Expression of PRP3-lacZ and PRP4-lacZ gene fusions is increased in spp41 strains, suggesting that wild-type Spp41p represses expression of PRP3 and PRP4. SPP41 was cloned and sequenced and found to be essential. spp43 is allelic to the previously identified suppressor srn1, which encodes a negative regulator of gene expression.     

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein.     

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.     

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.     

Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p.

The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the U1 snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive U1 snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the U1 snRNP and is recruited with the U1 snRNP into splicing complexes. On the basis of these and related observations, we propose that Prp39p functions, at least in part, prior to stable branch point recognition by the U1 snRNP particle to facilitate or stabilize the U1 snRNP/5' splice site interaction.     

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.     

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.     

The mammalian homologue of Prp16p is overexpressed in a cell line tolerant to Leflunomide, a new immunoregulatory drug effective against rheumatoid arthritis.

Prp2p, Prp16p, Prp22p, and Prp43p are members of the DEAH-box family of ATP-dependent putative RNA helicases required for pre-mRNA splicing in Saccharomyces cerevisiae. Recently, mammalian homologues of Prp43p and Prp22p have been described, supporting the idea that splicing in yeast and man is phylogenetically conserved. In this study, we show that a murine cell line resistant to the novel immunoregulatory drug Leflunomide (Arava) overexpresses a 135-kDa protein that is a putative DEAH-box RNA helicase. We have cloned the human counterpart of this protein and show that it shares pronounced sequence homology with Prp16p. Apart from its N-terminal domain, which is rich in RS, RD, and RE dipeptides, this human homologue of Prp16p (designated hPrp16p) is 41% identical to Prp16p. Significantly, homology is not only observed within the phylogenetically conserved helicase domain, but also in Prp16p-specific sequences. Immunofluorescence microscopy studies demonstrated that hPrp16p co-localizes with snRNPs in subnuclear structures referred to as speckles. Antibodies specific for hPrp16p inhibited pre-mRNA splicing in vitro prior to the second step. Thus, like its yeast counterpart, hPrp16p also appears to be required for the second catalytic step of splicing. Taken together, our data indicate that the human 135-kDa protein identified here is the structural and functional homologue of the yeast putative RNA helicase, Prp16p.     

Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex

PAP-1 has been identified by us as a Pim-1-binding protein and has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). We have then shown that PAP-1 plays a role in pre-mRNA splicing. Because four causative genes for adRP, including PAP-1, Prp31, Prp8, and Prp3, encode proteins that function as splicing factors or splicing-modulating factors, we investigated the interaction of PAP-1 with Prp3p and Prp31p in this study. The results showed that PAP-1 interacted with Prp3p but not Prp31p in human cells and yeast, and that the basic region of PAP-1 and the C-terminal region of Prp3p, regions beside spots found in adRP mutations, were needed for binding. Furthermore, both Prp3p and a part of PAP-1 were found to be components of the U4/U6.U5-tri-snRNP complex, one form of the spliceosome, in Ba/F3 and K562 cells by analysis of sucrose density gradients, suggesting that PAP-1 is weakly associated with the spliceosome. These results also suggest that splicing factors implicated in adRP contribute alone or mutually to proper splicing in the retina and that loss of their functions leads to onset of adRP.     

A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11).

We report mutations in a gene (PRPF31) homologous to Saccharomyces cerevisiae pre-mRNA splicing gene PRP31 in families with autosomal dominant retinitis pigmentosa linked to chromosome 19q13.4 (RP11; MIM 600138). A positional cloning approach supported by bioinformatics identified PRPF31 comprising 14 exons and encoding a protein of 499 amino acids. The level of sequence identity to the yeast PRP31 gene indicates that PRPF31 is also likely to be involved in pre-mRNA splicing. Mutations that include missense substitutions, deletions, and insertions have been identified in four RP11-linked families and three sporadic RP cases. The identification of mutations in a pre-mRNA splicing gene implicates defects in the splicing process as a novel mechanism of photoreceptor degeneration.     

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.     

Genetic interactions of conserved regions in the DEAD-box protein Prp28p.

The yeast PRP28 g ene has been implicated in nuclear precursor messenger RNA (pre-mRNA) splicing, a two-step reaction involved in a multitude of RNA structural alterations. Prp28p, the gene product of PRP28 , is a member of the evolutionarily conserved DEAD-box proteins (DBPs). Members of DBPs are involved in a variety of RNA-related biochemical processes, presumably by their putative RNA helicase activities. Prp28p has been speculated to play a role in melting the duplex between U4 and U6 small nuclear RNAs (snRNAs), leading to the formation of an active spliceosome. To study the function of Prp28p and its interactions with other components of the splicing machinery, we have isolated and characterized a large number of prp28 conditional mutants. Strikingly, many of these prp28 mutations are localized in the highly conserved motifs found in all the DBPs. Intragenic reversion analysis suggests that regions of motifs II, III and V, as well as of motifs I and IV, in Prp28p are likely to be in close proximity to each other. Our results thus provide the first hint of the local structural arrangement for Prp28p, and perhaps for other DBPs as well.     

Snt309p, a Component of the Prp19p-Associated Complex That Interacts with Prp19p and Associates with the Spliceosome Simultaneously with or Immediately after Dissociation of U4 in the Same Manner as Prp19p

The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y. Tarn, C.-H. Hsu, K.-T. Huang, H.-R. Chen, H.-Y. Kao, K.-R. Lee, and S.-C. Cheng, EMBO J. 13:2421–2431, 1994). To identify proteins in the Prp19p-associated complex, we have isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using the ade2-ade3 sectoring system. A novel splicing factor, Snt309p, was identified through such a screen. Although the SNT309 gene was not essential for growth of Saccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature. Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p. Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis. Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly. These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.     

Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor

We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G₁–S and G₂–M progression in the cell cycle. Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. Prp4p and Prp1p also interact in the yeast two-hybrid system. In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein. These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors.