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.     

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.     

Genetic Interactions between the Yeast RNA Helicase Homolog Prp16 and Spliceosomal Snrnas Identify Candidate Ligands for the Prp16 RNA-Dependent Atpase

Pre-mRNA splicing occurs in a large and dynamic ribonucleoprotein complex, the spliceosome. Several protein factors involved in splicing are homologous to a family of RNA-dependent ATPases, the so-called DEAD/DEAH proteins. A subset of these factors exhibit RNA helicase activity in vitro. The DEAD/DEAH proteins involved in splicing are thought to mediate RNA conformational rearrangements during spliceosome assembly. However, the RNA ligands for these factors are currently unknown. Here, we present genetic evidence in Saccharomyces cerevisiae for a functional interaction between the DEAH protein Prp16, and the U6 and U2 spliceosomal snRNAs. Using a library of mutagenized U6 snRNA genes, we have identified 14 strong suppressors of the cold-sensitive (cs) allele, prp16-302. Remarkably, each suppressor contains a single nucleotide deletion of 1 of the 6 residues that lie immediately upstream of a sequence in U6 that interacts with the 5' splice site. Analysis of site-directed mutations revealed that nucleotide substitutions in the adjacent U2-U6 helix I structure also suppress prp16-302, albeit more weakly. The U6 suppressors tested also partially reverse the phenotype of two other cs alleles, prp16-1 and prp16-301, but not the four temperature-sensitive alleles tested. Finally, overexpression of each cs allele exacerbates its recessive growth phenotype and confers a dominant negative cs phenotype. We propose that the snRNA suppressors function by destabilizing an interaction between the U2-U6 complex and a hypothetical factor (X), which is trapped by cs mutants of PRP16. The phenotypes of overexpressed prp16 alleles are consistent with the model that this trapped interaction inhibits the dissociation of Prp16 from the spliceosome. We discuss the intriguing possibility that factor X is Prp16 itself.     

Genetic studies of the PRP11 gene of Saccharomyces cerevisiae

Summary PRP11 is a gene that encodes an essential function for pre-messenger RNA (mRNA) processing in Saccharomyces cerevisiae. We have carried out a mutational study to locate essential and non-essential regions of the PRP11 protein. The existing temperature-sensitive (ts) mutation (prpl1-1) was isolated from the chromosome of the original mutant and its position in the gene was determined. When the prp11-1 gene was transcribed from the GAL1 promoter, the overproduced protein was able to reverse the is prp11-1 phenotype; this is compatible with the possibility that the defect in the prp11-1 gene product affects its binding to the spliceosome. Thirteen linker-insertion mutations were constructed. Only five (prp11-4, 11-6, 11-10, –13 and –14) resulted in a null phenotype. One of these became temperature-sensitive when the insertion was.reduced in size from four (prp11-10) to two (prp11-15) amino acids. A sequence of ten amino acids of which also occurs in the human U1 small nuclear ribonucleoprotein particle (snRNP) A protein and the U2 snRNP B protein, when deleted from PRP11, had no phenotype and thus appears to be nonessential for PRP11 function. However, a linker-insertion mutation (prp11–10) immediately adjacent to this region resulted in a null phenotype.     

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.     

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 mutation in a methionine tRNA gene suppresses the prp2-1 Ts mutation and causes a pre-mRNA splicing defect in Saccharomyces cerevisiae.

The PRP2 gene in Saccharomyces cerevisiae encodes an RNA-dependent ATPase that activates spliceosomes for the first transesterification reaction in pre-mRNA splicing. We have identified a mutation in the elongation methionine tRNA gene EMT1 as a dominant, allele-specific suppressor of the temperature-sensitive prp2-1 mutation. The EMT1-201 mutant suppressed prp2-1 by relieving the splicing block at high temperature. Furthermore, EMT1-201 single mutant cells displayed pre-mRNA splicing and cold-sensitive growth defects at 18 degrees. The mutation in EMT1-201 is located in the anticodon, changing CAT to CAG, which presumably allowed EMT1-201 suppressor tRNA to recognize CUG leucine codons instead of AUG methionine codons. Interestingly, the prp2-1 allele contains a point mutation that changes glycine to aspartate, indicating that EMT1-201 does not act by classical missense suppression. Extra copies of the tRNA(Leu)(UAG) gene rescued the cold sensitivity and in vitro splicing defect of EMT1-201. This study provides the first example in which a mutation in a tRNA gene confers a pre-mRNA processing (prp) phenotype.     

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.     

The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP.

We have raised specific antibodies against the PRP6 protein and shown that the U4, U5 and U6 snRNAs are co-precipitated with this protein. Using splicing extracts prepared from in vivo heat-inactivated cells, we have characterized the prp4-1 and prp6-1 biochemical defects. In inactivated prp4-1 cell extracts, the U6 snRNA content as well as the U6, U4/U6 snRNPs and the [U4/U6.U5] tri-snRNP particles amounts are severely reduced. In inactivated prp6-1 cell extracts, the PRP6 mutant protein is barely detectable. Glycerol gradient analyses indicate that, in these extracts, the [U4/U6.U5] tri-snRNPs are present in very low amounts, but U4/U6 snRNP particles are normally represented. These results establish that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. We found no evidence for the presence of the PRP6 protein in the U4/U6 particle.     

The molecular characterization of PRP6 and PRP9 yeast genes reveals a new cysteine/histidine motif common to several splicing factors.

prp6 and prp9 thermosensitive (ts) mutants are affected in pre-mRNA splicing and transport from the nucleus to the cytoplasm. PRP6 and PRP9 wild-type alleles have been sequenced. DNA sequence analysis reveals homologies in the 5' and 3' non-coding regions, suggesting a common regulation of gene expression. PRP6 and PRP9 genes encode a 899 amino acid and a 530 amino acid protein, respectively. The PRP6 protein has repeated motifs that evoke helix-loop-helix structures. Both PRP6 and PRP9 proteins have cysteine/histidine motifs loosely related to those found in zinc finger proteins. The substitution of some, but not all, of these residues by directed mutagenesis has a critical effect on the protein function. Homology searches reveal that two other proteins known to be involved in the nuclear splicing pathway--the yeast PRP11 and the human U1C proteins--contain similar sequences. The five cysteine/histidine motifs found in these four proteins display amino acid similarities in addition to the cysteine and histidine residues, indicating that they participate in biological structures or functions related to the splicing process. In addition, PRP6 and PRP9 exhibit leucine repeat motifs which may be implicated in protein interactions. The prp6 and prp9 ts mutations have been mapped and sequenced.     

The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.

PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.     

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.     

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.     

Allele-specific suppression of aSaccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search forprp20-10 allele-specific high-copy-number suppressors, theKSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.     

Allele-specific suppression of aSaccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search forprp20-10 allele-specific high-copy-number suppressors, theKSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.     

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.