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.     

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.     

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.     

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.     

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 PRP4 (RNA4) protein of Saccharomyces cerevisiae is associated with the 5'' portion of the U4 small nuclear RNA.

We have combined oligonucleotide-directed RNase H degradation and immunoprecipitation in a study of the association of the Saccharomyces cerevisiae PRP4 protein with the U4-U6 complex. We have found that three oligonucleotides were able to direct nearly to completion the RNase H-specific cleavage of the target RNA molecules as they exist in splicing extracts. Immunoprecipitation of the degradation products with PRP4 antibody showed that the 5' portion of U4 small nuclear RNA (snRNA) and the 3' portion of U6 snRNA coimmunoprecipitated with the PRP4 protein. Micrococcal nuclease protection experiments confirmed further that the 5' portion and 3' end of U4 snRNA were very resistant to nuclease digestion, whereas the 3' portion of U6 snRNA was protected to only a very small extent. We conclude that the PRP4 protein of S. cerevisiae is associated primarily with the 5' portion of U4 snRNA in the U4-U6 small nuclear ribonucleoprotein (snRNP).     

PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro.

We previously reported the isolation of PRP28, a gene in Saccharomyces cerevisiae whose activity is required for the first step of nuclear mRNA splicing in vivo. Sequence analysis revealed that PRP28 is included in the 'DEAD-box' gene family, members of which are thought to function as ATP-dependent RNA helicases. Genetic interactions led us to suggest that PRP28 is functionally associated with the U4/U5/U6 snRNP. We have now purified the PRP28 protein from S. cerevisiae and demonstrated that it is required for the first step of splicing in vitro. Interestingly, PRP28 is not a stably associated snRNP protein. Strand displacement assays indicate that PRP28 does not exhibit RNA helicase activity, suggesting that an additional factor or factors may be required for its activation.     

The nuclear matrix phosphoprotein p255 associates with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle.

The monoclonal antibody CC3 recognizes a phosphorylated epitope present on an interphase protein of 255 kDa. Previous work has shown that p255 is localized mainly to nuclear speckles and remains associated with the nuclear matrix scaffold following extraction with non-ionic detergents, nucleases and high salt. The association of p255 with splicing complexes is suggested by the finding that mAb CC3 can inhibit in vitro splicing and immunoprecipitate pre-messenger RNA and splicing products. Small nuclear RNA immunoprecipitation assays show that p255 is a component of the U5 small nuclear ribonucleoprotein (snRNP) and the [U4/U6.U5] tri-snRNP complex. In RNase protection assays, mAb CC3 immunoprecipitates fragments containing branch site and 3' splice site sequences. As predicted for a [U4/U6.U5]-associated component, the recovery of the branch site-protected fragment requires binding of U2 snRNP and is inhibited by EDTA. p255 may correspond to the previously identified p220 protein, the mammalian analogue of the yeast PRP8 protein. Our results suggest that changes in the phosphorylation of p255 may be part of control mechanisms that interface splicing activity with nuclear organization.     

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.     

U4 and U6 small nuclear ribonucleoprotein particles. 2. Evidence for their involvement in pre-mRNA splicing

The in vitro splicing of pre-mRNA of the human beta-globin gene in the presence of HeLa cell nuclear extract was investigated. Splicing was inhibited by auto-antibodies against U4 and U6 snRNP particles. No intermediates or products of the splicing reaction were evident in the presence of antibodies against U4 and U6 snRNPs which suggests their involvement in pre-mRNA splicing.     

The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase.

Unlike autocatalyzed self-splicing reactions, nuclear pre-mRNA splicing requires transacting macromolecules and ATP. A protein encoded by the PRP2 gene of Saccharomyces cerevisiae is required, in conjunction with ATP, for the first cleavage-ligation reaction of pre-mRNA splicing. In this study, we have purified two forms of the PRP2 gene product with apparent molecular weights of 100 kDa and 92 kDa, from a yeast strain overproducing the protein. Both proteins were indistinguishable in their ability to complement extracts derived from a heat-sensitive prp2 mutant. Furthermore, we show that the PRP2 protein is capable of hydrolyzing nucleoside triphosphates in the presence of single-stranded RNAs such as poly(U). However, purified PRP2 by itself did not unwind double-stranded RNA substrates. The fact that an RNA-dependent NTPase activity is intrinsic to PRP2 may account for the ATP requirement in the first catalytic reaction of pre-mRNA splicing.     

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.     

Developmental and tissue-specific expression of U4 small nuclear RNA genes.

U4 RNA is one of several small nuclear RNAs involved in the splicing of mRNA precursors. The domestic chicken has two genes per haploid genome that are capable of encoding U4 RNA. The U4X RNA gene (which encodes a sequence variant of U4 RNA that was unknown prior to the cloning of the gene) and the U4B RNA gene were both expressed in vivo in each of seven adult and three embryonic chicken tissues examined. However, the ratio of U4B RNA to U4X RNA can vary more than sevenfold in both a tissue- and stage-specific manner.     

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.     

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.