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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2959-2970.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2959-2970

Author(s):

D S Horowitz ◽

J Abelson

Keyword(s):

Saccharomyces Cerevisiae ◽

Active Form ◽

Mrna Splicing ◽

Second Step ◽

Temperature Sensitive ◽

Ribonucleoprotein Particle ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2959 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2959-2970 ◽

Cited By ~ 32

Author(s):

D S Horowitz ◽

J Abelson

Keyword(s):

Saccharomyces Cerevisiae ◽

Active Form ◽

Mrna Splicing ◽

Second Step ◽

Temperature Sensitive ◽

Ribonucleoprotein Particle ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

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.

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PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle.

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3698 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3698-3709 ◽

Cited By ~ 48

Author(s):

S P Bjørn ◽

A Soltyk ◽

J D Beggs ◽

J D Friesen

Keyword(s):

Gene Product ◽

Yeast Cells ◽

Temperature Sensitive ◽

Ribonucleoprotein Particle ◽

Direct Role ◽

Small Nuclear Ribonucleoprotein ◽

Cloned Gene ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

The PRP4 (RNA4) gene product is involved in nuclear mRNA processing in yeast cells; we have previously cloned the gene by complementation of a temperature-sensitive mutation. Sequence and transcript analyses of the cloned gene predicted the gene product to be a 52-kilodalton protein, which was confirmed with antibodies raised against the PRP4 gene product. These antibodies inhibited precursor mRNA splicing in vitro, demonstrating a direct role of PRP4 in splicing. Immunoprecipitations with the antibodies indicated that the PRP4 protein is associated with the U4/U6 small nuclear ribonucleoprotein particle.

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Identification of Prp40, a novel essential yeast splicing factor associated with the U1 small nuclear ribonucleoprotein particle.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.960 ◽

1996 ◽

Vol 16 (3) ◽

pp. 960-967 ◽

Cited By ~ 54

Author(s):

H Y Kao ◽

P G Siliciano

Keyword(s):

Essential Gene ◽

Temperature Sensitive ◽

Small Nuclear Rna ◽

Ribonucleoprotein Particle ◽

Direct Role ◽

Small Nuclear Ribonucleoprotein ◽

Cold Sensitive ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

We have used suppressor genetics to identify factors that interact with Saccharomyces cerevisiae U1 small nuclear RNA (snRNA). In this way, we isolated PRP40-1, a suppressor that restores growth at 18 degrees C to a strain bearing a cold-sensitive mutation in U1 RNA. A gene disruption experiment shows that PRP40 is an essential gene. To study the role of PRP40 in splicing, we created a pool of temperature-sensitive prp40 strains. Primer extension analysis of intron-containing transcripts in prp40 temperature-sensitive strains reveals a splicing defect, indicating that Prp40 plays a direct role in pre-mRNA splicing. In addition, U1 RNA coimmunoprecipitates with Pro40, indicating that Prp40 is bound to the U1 small nuclear ribonucleoprotein particle in vivo. Therefore, we conclude that PRP40 encodes a novel, essential splicing component that associates with the yeast U1 small nuclear ribonucleoprotein particle.

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PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3698-3709.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3698-3709

Author(s):

S P Bjørn ◽

A Soltyk ◽

J D Beggs ◽

J D Friesen

Keyword(s):

Gene Product ◽

Yeast Cells ◽

Temperature Sensitive ◽

Ribonucleoprotein Particle ◽

Direct Role ◽

Small Nuclear Ribonucleoprotein ◽

Cloned Gene ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

The PRP4 (RNA4) gene product is involved in nuclear mRNA processing in yeast cells; we have previously cloned the gene by complementation of a temperature-sensitive mutation. Sequence and transcript analyses of the cloned gene predicted the gene product to be a 52-kilodalton protein, which was confirmed with antibodies raised against the PRP4 gene product. These antibodies inhibited precursor mRNA splicing in vitro, demonstrating a direct role of PRP4 in splicing. Immunoprecipitations with the antibodies indicated that the PRP4 protein is associated with the U4/U6 small nuclear ribonucleoprotein particle.

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Sex-lethal interacts with splicing factors in vitro and in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.5036 ◽

1996 ◽

Vol 16 (9) ◽

pp. 5036-5047 ◽

Cited By ~ 25

Author(s):

G Deshpande ◽

M E Samuels ◽

P D Schedl

Keyword(s):

Rna Splicing ◽

Target Genes ◽

Ribonucleoprotein Particle ◽

Downstream Target ◽

Small Nuclear Ribonucleoprotein ◽

Sex Lethal ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

The Drosophila sex determination gene Sex-lethal controls its own expression and the expression of downstream target genes such as transformer by regulating RNA splicing. Genetic and molecular studies have established that Sxl requires the product of another gene, snf, to autoregulate the splicing of its own transcripts. snf has recently been shown to encode a Drosophila U1 and U2 small nuclear ribonucleoprotein particle protein. In the work reported here, we demonstrate that the Sxl and Snf proteins can interact directly in vitro and that these two proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with the Sxl antibody. Unlike bulk Snf protein, which sediments slowly in sucrose gradients, the Snf protein associated with Sxl is in a large, rapidly sedimenting complex. Detailed characterization of the Sxl-Snf complexes from cross-linked extracts indicates that these complexes contain additional small nuclear ribonucleoprotein particle proteins and the U1 and U2 small nuclear RNAs. Finally, consistent with the RNase sensitivity of the Sxl-Snf complexes, Sxl transcripts can also be immunoprecipitated by Sxl antibodies. On the basis of the physical interactions between Sxl and Snf, we present a model for Sxl splicing regulation. This model helps explain how the Sxl protein is able to promote the sex-specific splicing of Sxl transcripts, utilizing target sequences that are distant from the regulated splice sites.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3710-3719.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3710-3719

Author(s):

J Banroques ◽

J N Abelson

Keyword(s):

Essential Role ◽

Mrna Splicing ◽

Ribonucleoprotein Particle ◽

Specific Antibodies ◽

Small Nuclear Ribonucleoprotein ◽

Assembly Pathway ◽

Nuclear Ribonucleoprotein ◽

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.

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Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1258-1269.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1258-1269

Author(s):

M Himmelspach ◽

R Gattoni ◽

C Gerst ◽

K Chebli ◽

J Stévenin

Keyword(s):

Donor Site ◽

Ribonucleoprotein Particle ◽

Adenovirus E1a ◽

Branch Site ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

U1 Snrnp ◽

The Common ◽

Nuclear Ribonucleoprotein

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

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Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.445 ◽

1995 ◽

Vol 15 (1) ◽

pp. 445-455 ◽

Cited By ~ 30

Author(s):

J Roy ◽

B Zheng ◽

B C Rymond ◽

J L Woolford

Keyword(s):

Protein Complexes ◽

Mrna Splicing ◽

Yeast Cells ◽

Ribonucleoprotein Particle ◽

Sm Proteins ◽

Small Nuclear Ribonucleoprotein ◽

Snrnp Protein ◽

Nuclear Ribonucleoprotein ◽

Precursor Mrna

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.

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