PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.     

A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced. Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently. This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing. The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity. Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts. From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA. PRP18 therefore appears to be primarily associated with the U5 snRNP. The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro. Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.     

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 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.     

PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.     

Splicing factor SF4 is dispensable for the assembly of a functional splicing complex and participates in the subsequent steps of the splicing reaction.

The splicing of nuclear messenger RNA precursors (pre-mRNA) can be reconstituted in vitro with factors partially purified from HeLa cell nuclear extracts. Splicing complexes are assembled in the presence of the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 and the protein factors SF1, SF2, SF3 and U2AF. However, the complexes thus formed are inactive, i.e. they only contain unprocessed pre-mRNA. The intermediates and products of the splicing reaction are generated after addition of SF4. This splicing factor is a heat-labile protein which requires sulfhydryl groups for its activity. SF4 appears to participate, directly or indirectly, in the conversion of a functional but inactive splicing complex to the active spliceosome.     

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.     

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.     

Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing.

To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA. We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA. SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 [U. Vijayraghavan, M. Company, and J. Abelson, Genes Dev. 3:1206-1216, 1989]) are required only for the second step of splicing. Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4. We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing.     

Sequence-specific affinity selection of mammalian splicing complexes.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/biotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.     

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.     

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.     

U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.     

Roles of U4 and U6 snRNAs in the assembly of splicing complexes.

A series of U4 and U6 snRNA mutants was analysed in Xenopus oocytes to determine whether they block splicing complex assembly or splicing itself. All the U4 and U6 mutants found to be inactive in splicing complementation resulted in defects in assembly of either U4/U6 snRNP or of splicing complexes. No mutants were found to separate the entry of U5 and U6 snRNAs into splicing complexes and neither of these RNAs was able to associate with the pre-mRNA in the absence of U4. In the absence of U6 snRNA, however, U4 entered a complex containing pre-mRNA as well as the U1 and U2 snRNAs. U6 nucleotides whose mutation resulted in specific blockage of the second step of splicing in Saccharomyces cerevisiae are shown not to be essential for splicing in the oocyte assay. The results are discussed in terms of the roles of U4 and U6 in the assembly and catalytic steps of the splicing process.     

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.     

Interactions of the yeast U6 RNA with the pre-mRNA branch site.

The small nuclear RNA (snRNA) components of the spliceosome have been proposed to catalyze the excision of introns from nuclear pre-mRNAs. If this hypothesis is correct, then the snRNA components of the spliceosome may interact directly with the reactive groups of pre-mRNA substrates. To explore this possibility, a genetic screen has been used to identify potential interactions between the U6 RNA and the pre-mRNA branch site. Notably, the selection yielded mutants in two regions of the yeast U6 RNA implicated previously in the catalytic events of splicing. These mutants significantly increase the splicing of pre-mRNA substrates containing non-adenosine branch sites. U6 mutants in U2/U6 helix Ia show strong allele-specific interactions with the branch site nucleotide and interact with PRP16, a factor implicated previously in branch site utilization. The other mutants cluster in the intramolecular helix of U6 and suppress the effects of branch site mutations in a nonallele-specific fashion. The locations of these mutants may define positions important for binding of the U6 intramolecular helix to the catalytic core of the spliceosome.