A novel gene, spp91-1, suppresses the splicing defect and the pre-mRNA nuclear export in the prp9-1 mutant.

Processing and export of nuclear pre-mRNA are believed to be competing processes in the nucleus. In order to identify factors which are involved in these processes, we isolated suppressors that relieve the growth defect of a prp9-1 temperature-sensitive mutant strain of Saccharomyces cerevisiae. The prp9-1 mutation was previously shown to abolish splicing and to target pre-mRNA to the cytoplasm. One of the suppressors, spp91-1, corrects the prp9-1 growth defect through partial restoration of splicing and by a complete reversion of the pre-mRNA escape phenotype. This suppressor is specific for two prp9 alleles and cannot substitute for PRP9 function. The mutant and wild-type alleles of SPP91 were cloned and sequenced. SPP91 encodes a novel protein essential for mitotic growth whose sequence contains motifs indicative of a nuclear localization. In vivo depletion of SPP91 in a prp9-1 genetic background is lethal and is associated with reduced amounts of spliced mRNA and accumulation of pre-mRNA. This observation strongly supports the hypothesis that SPP91 encodes a PRP factor. We suggest that spp91-1 increases pre-mRNA retention in the nucleus by improving the formation of the spliceosome and thereby allowing a larger proportion of the pre-mRNA molecules to be spliced.     

An extragenic suppressor ofprp24-1 defines genetic interaction betweenPRP24 andPRP21 gene products ofSaccharomyces cerevisiae

The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.     

An extragenic suppressor ofprp24-1 defines genetic interaction betweenPRP24 andPRP21 gene products ofSaccharomyces cerevisiae

The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.     

Six novel genes necessary for pre-mRNA splicing in Saccharomyces cerevisiae.

We have identified six new genes whose products are necessary for the splicing of nuclear pre-mRNA in the yeast Saccharomyces cerevisiae. A collection of 426 temperature-sensitive yeast strains was generated by EMS mutagenesis. These mutants were screened for pre-mRNA splicing defects by an RNA gel blot assay, using the intron- containing CRY1 and ACT1 genes as hybridization probes. We identified 20 temperature-sensitive mutants defective in pre-mRNA splicing. Twelve appear to be allelic to the previously identified prp2, prp3, prp6, prp16/prp23, prp18, prp19 or prp26 mutations that cause defects in spliceosome assembly or the first or second step of splicing. One is allelic to SNR14 encoding U4 snRNA. Six new complementation groups, prp29-prp34, were identified. Each of these mutants accumulates unspliced pre-mRNA at 37 degrees C and thus is blocked in spliceosome assembly or early steps of pre-mRNA splicing before the first cleavage and ligation reaction. The prp29 mutation is suppressed by multicopy PRP2 and displays incomplete patterns of complementation with prp2 alleles, suggesting that the PRP29 gene product may interact with that of PRP2. There are now at least 42 different gene products, including the five spliceosomal snRNAs and 37 different proteins that are necessary for pre-mRNA splicing in Saccharomyces cerevisiae. However, the number of yeast genes identifiable by this approach has not yet been exhausted.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

spp42, identified as a classical suppressor of prp4-73, which encodes a kinase involved in pre-mRNA splicing in fission yeast, is a homologue of the splicing factor Prp8p.

We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4(+) gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73(ts) at the restrictive temperature (36 degrees ). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73(ts), indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73(ts) mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes.     

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.     

A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.     

A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome.

PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.     

Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors.

The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process.     

An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs

The screening of antisuppressor mutants of the yeast Schizosaccharomyces pombe has been successfully accomplished with high resolution liquid chromatographic methods for the analysis of tRNA nucleosides. Antisuppressor mutations reduce or abolish the function of nonsense suppressor-tRNAs or other informational suppressors. Nonradioactive or 35S-labeled unfractionated tRNA from various strains was digested to nucleosides and analyzed by high performance liquid chromatography. The mutant sin3 has lost the nucleoside 5-(methoxycarbonylmethyl)-2-thiouridine from its tRNA in comparison to parental strains. In eukaryotes this nucleoside is found at the first position of the anticodon (wobble position) in several isoacceptor tRNAs that preferentially recognize codons ending with adenosine. The sin3 mutation reduces the efficiency of UGA and UAA suppressor tRNASer and suppressor tRNALeu. The genetic cosegregation of modification loss, antisuppressor phenotype, and a change in cell size is demonstrated. This indicates that a single mutation in the structural gene for a tRNA modification enzyme causes the three different phenotypes.     

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.     

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.     

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.     

Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing

Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing. Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43. Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases. Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity. Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process. First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins. SPP2 was originally isolated as a genetic suppressor of a prp2 mutant. In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2. Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction. A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain. The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.     

The fission yeast prp4+ gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth.

Only four prp (pre-mRNA processing) genes of the fission yeast Schizosaccharomyces pombe have been reported. We exploited yeast genetics and identified and isolated the prp4 gene. Sequence analysis revealed that the splicing factor encoded by this gene contains the signature sequences that define the serine/threonine protein kinase family. This is the first kinase gene identified whose product is involved in pre-mRNA splicing. The prp4 gene contains one intron in the kinase domain. Gene replacement studies provided evidence that this gene is essential for growth and is located on chromosome III.     

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.