ABSTRACT
Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing. No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts. Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP. Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs. Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions. Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly. These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs. Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs. Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans.
Subject(s)
Fungal Proteins/metabolism , Nuclear Proteins/metabolism , RNA Splicing , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Blotting, Northern , Centrifugation, Density Gradient , DNA Probes/chemistry , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/genetics , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nucleic Acid Conformation , Precipitin Tests , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/chemistry , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/genetics , Saccharomyces cerevisiae/genetics , Sodium Chloride/pharmacology , Spliceosomes/metabolismABSTRACT
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.
Subject(s)
Fungal Proteins/metabolism , RNA Splicing , Ribonucleoprotein, U4-U6 Small Nuclear/ultrastructure , Ribonucleoprotein, U5 Small Nuclear/ultrastructure , Saccharomyces cerevisiae Proteins , Spliceosomes/ultrastructure , Macromolecular Substances , Nucleic Acid Precursors/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA, Small Nuclear/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoprotein, U5 Small Nuclear/metabolism , Saccharomyces cerevisiae , Spliceosomes/metabolismABSTRACT
The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.
Subject(s)
Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal , RNA Precursors/metabolism , RNA, Fungal/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cloning, Molecular , Mutation , RNA Processing, Post-Transcriptional , RNA Splicing/genetics , Restriction Mapping , TemperatureABSTRACT
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 suppressors 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.
