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

1996 ◽  
Vol 16 (3) ◽  
pp. 960-967 ◽  
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.

scholarly journals 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.

1990 ◽  
Vol 10 (12) ◽  
pp. 6417-6425 ◽  
Author(s):  
N Abovich ◽  
P Legrain ◽  
M Rosbash
Keyword(s):  
Temperature Sensitive ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Snrnp Protein ◽  
Yeast Genes ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

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.

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

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.

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

1990 ◽  
Vol 10 (12) ◽  
pp. 6417-6425
Author(s):  
N Abovich ◽  
P Legrain ◽  
M Rosbash
Keyword(s):  
Temperature Sensitive ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Snrnp Protein ◽  
Yeast Genes ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

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.

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

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.

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

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.

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

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.

scholarly journals The amino-terminal domain of yeast U1-70K is necessary and sufficient for function.

1995 ◽  
Vol 15 (11) ◽  
pp. 6341-6350 ◽  
Author(s):  
P J Hilleren ◽  
H Y Kao ◽  
P G Siliciano
Keyword(s):  
Rna Recognition Motif ◽  
Ribonucleoprotein Particle ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
Necessary And Sufficient ◽  
Nuclear Ribonucleoprotein ◽  
Amino Terminal Domain ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation.

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

1996 ◽  
Vol 16 (9) ◽  
pp. 5036-5047 ◽  
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.

Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein

1990 ◽  
Vol 10 (9) ◽  
pp. 4480-4485
Author(s):  
J Andersen ◽  
R J Feeney ◽  
G W Zieve
Keyword(s):  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Identification And Characterization ◽  
Snrnp Core ◽  
Small Nuclear Ribonucleoprotein Particle

The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera.

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