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Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19.

Ren L, McLean JR, Hazbun TR, Fields S, Vander Kooi C, Ohi MD, Gould KL - PLoS ONE (2011)

Bottom Line: Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation.The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation.Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Vanderbilt University, Nashville, Tennessee, [corrected] United States of America.

ABSTRACT
Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

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ScUrn1/SpDre4 is involved in pre-mRNA splicing.A) RNA was purified from the indicated S. pombe strains grown at 25°C(−) or shifted to 36°C (+) for 4 hours. RT-PCR reactions were performed using oligonucleotides that flank the long intron within the prp19 mRNA. PCR products were separated on 3% Nusieve gels and detected with ethidium-bromide and UV imaging. B) Northern analysis of RNA isolated from an anti-cap or anti-GFP immunoprecipitate from wild-type cells, or RNA isolated from the indicated TAP purifications. Each RNA sample was probed for the presence of the U1, U2, U4, U5, and U6 snRNAs. C–E) Prp19-TAP complexes were isolated from the indicated S. pombe strains and a portion of the eluates was probed for the presence of the indicated proteins. The remainder of the eluates were divided in half. One half was immunoprecipitated with anti-Myc and the other with anti-GFP and then each immunoprecipitate was immunoblotted with anti-GFP or anti-Myc antibodies.
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pone-0016719-g005: ScUrn1/SpDre4 is involved in pre-mRNA splicing.A) RNA was purified from the indicated S. pombe strains grown at 25°C(−) or shifted to 36°C (+) for 4 hours. RT-PCR reactions were performed using oligonucleotides that flank the long intron within the prp19 mRNA. PCR products were separated on 3% Nusieve gels and detected with ethidium-bromide and UV imaging. B) Northern analysis of RNA isolated from an anti-cap or anti-GFP immunoprecipitate from wild-type cells, or RNA isolated from the indicated TAP purifications. Each RNA sample was probed for the presence of the U1, U2, U4, U5, and U6 snRNAs. C–E) Prp19-TAP complexes were isolated from the indicated S. pombe strains and a portion of the eluates was probed for the presence of the indicated proteins. The remainder of the eluates were divided in half. One half was immunoprecipitated with anti-Myc and the other with anti-GFP and then each immunoprecipitate was immunoblotted with anti-GFP or anti-Myc antibodies.

Mentions: While Prp17 and ScCwc2/SpCwf2 are known splicing factors, a role for ScUrn1/SpDre4 in pre-mRNA splicing has not been described previously. We obtained multiple lines of evidence that ScUrn1/SpDre4 impacts this process. First, S. pombe dre4Δ cells are viable but temperature-sensitive for growth (Figure S2B; [56]). At the non-permissive temperature of 36°C, they accumulated prp19 pre-mRNA, indicative of defective pre-mRNA splicing, whereas wild type cells did not (Figure 5A). Second, like prp17Δ, dre4Δ is synthetically lethal with the cdc5-120 splicing mutation at 25°C; in 12 and 11 tetrads, respectively, no viable Ura+ Ts recombinants were obtained. Similarly, S. cerevisiae urn1Δ has been reported to interact negatively with a variety of splicing mutations in global synthetic genetic array screens [57], [58]. Third, SpDre4 amino acids 1–300, which contain the WW and FF domains (Prp19-interacting region), were sufficient to rescue the S. pombe dre4Δ strain at 36°C whereas a truncation expressing only the WW domain (amino acids 1–183) very weakly supported growth and failed to promote wild-type morphology (Figure S2C and data not shown). These data suggest that interaction with Prp19 is critical for SpDre4 function.


Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19.

Ren L, McLean JR, Hazbun TR, Fields S, Vander Kooi C, Ohi MD, Gould KL - PLoS ONE (2011)

ScUrn1/SpDre4 is involved in pre-mRNA splicing.A) RNA was purified from the indicated S. pombe strains grown at 25°C(−) or shifted to 36°C (+) for 4 hours. RT-PCR reactions were performed using oligonucleotides that flank the long intron within the prp19 mRNA. PCR products were separated on 3% Nusieve gels and detected with ethidium-bromide and UV imaging. B) Northern analysis of RNA isolated from an anti-cap or anti-GFP immunoprecipitate from wild-type cells, or RNA isolated from the indicated TAP purifications. Each RNA sample was probed for the presence of the U1, U2, U4, U5, and U6 snRNAs. C–E) Prp19-TAP complexes were isolated from the indicated S. pombe strains and a portion of the eluates was probed for the presence of the indicated proteins. The remainder of the eluates were divided in half. One half was immunoprecipitated with anti-Myc and the other with anti-GFP and then each immunoprecipitate was immunoblotted with anti-GFP or anti-Myc antibodies.
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Related In: Results  -  Collection

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pone-0016719-g005: ScUrn1/SpDre4 is involved in pre-mRNA splicing.A) RNA was purified from the indicated S. pombe strains grown at 25°C(−) or shifted to 36°C (+) for 4 hours. RT-PCR reactions were performed using oligonucleotides that flank the long intron within the prp19 mRNA. PCR products were separated on 3% Nusieve gels and detected with ethidium-bromide and UV imaging. B) Northern analysis of RNA isolated from an anti-cap or anti-GFP immunoprecipitate from wild-type cells, or RNA isolated from the indicated TAP purifications. Each RNA sample was probed for the presence of the U1, U2, U4, U5, and U6 snRNAs. C–E) Prp19-TAP complexes were isolated from the indicated S. pombe strains and a portion of the eluates was probed for the presence of the indicated proteins. The remainder of the eluates were divided in half. One half was immunoprecipitated with anti-Myc and the other with anti-GFP and then each immunoprecipitate was immunoblotted with anti-GFP or anti-Myc antibodies.
Mentions: While Prp17 and ScCwc2/SpCwf2 are known splicing factors, a role for ScUrn1/SpDre4 in pre-mRNA splicing has not been described previously. We obtained multiple lines of evidence that ScUrn1/SpDre4 impacts this process. First, S. pombe dre4Δ cells are viable but temperature-sensitive for growth (Figure S2B; [56]). At the non-permissive temperature of 36°C, they accumulated prp19 pre-mRNA, indicative of defective pre-mRNA splicing, whereas wild type cells did not (Figure 5A). Second, like prp17Δ, dre4Δ is synthetically lethal with the cdc5-120 splicing mutation at 25°C; in 12 and 11 tetrads, respectively, no viable Ura+ Ts recombinants were obtained. Similarly, S. cerevisiae urn1Δ has been reported to interact negatively with a variety of splicing mutations in global synthetic genetic array screens [57], [58]. Third, SpDre4 amino acids 1–300, which contain the WW and FF domains (Prp19-interacting region), were sufficient to rescue the S. pombe dre4Δ strain at 36°C whereas a truncation expressing only the WW domain (amino acids 1–183) very weakly supported growth and failed to promote wild-type morphology (Figure S2C and data not shown). These data suggest that interaction with Prp19 is critical for SpDre4 function.

Bottom Line: Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation.The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation.Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Vanderbilt University, Nashville, Tennessee, [corrected] United States of America.

ABSTRACT
Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

Show MeSH