Limits...
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.

Show MeSH
Characterization of S. pombe Safs.A) Protein G pull-downs from the indicated S. pombe strains were blotted with antibodies to the HA epitope. The bands with asterisks correspond to the indicated proteins and were quantified relative to background. B and C) Spores from the (B) saf2::ura4+/saf2+ and (D) saf3::ura4+/saf3+ diploids were germinated in minimal medium lacking uracil. Cells were fixed in formaldehyde at 15 and 40 h, respectively, and stained with DAPI. D and E) RNA was purified from wildtype cells grown at 32°C, prp2-1 cells grown at 25°C(−) or shifted to 36°C (+) for 4 hours, or from spores germinated at 32°C for 24 h from saf2::ura4+/saf2+ (D) and saf3::ura4+/saf3+ (E) diploids in medium lacking uracil. 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. Arrows indicate the position of prescursor and mature RNA species.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046128&req=5

pone-0016719-g008: Characterization of S. pombe Safs.A) Protein G pull-downs from the indicated S. pombe strains were blotted with antibodies to the HA epitope. The bands with asterisks correspond to the indicated proteins and were quantified relative to background. B and C) Spores from the (B) saf2::ura4+/saf2+ and (D) saf3::ura4+/saf3+ diploids were germinated in minimal medium lacking uracil. Cells were fixed in formaldehyde at 15 and 40 h, respectively, and stained with DAPI. D and E) RNA was purified from wildtype cells grown at 32°C, prp2-1 cells grown at 25°C(−) or shifted to 36°C (+) for 4 hours, or from spores germinated at 32°C for 24 h from saf2::ura4+/saf2+ (D) and saf3::ura4+/saf3+ (E) diploids in medium lacking uracil. 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. Arrows indicate the position of prescursor and mature RNA species.

Mentions: Two other predicted proteins were identified in the S. pombe NTC component TAPs discussed above with high sequence coverage (Figure 3 and Tables S2 and S3). Encoded by ORFs SPAC2F3.14c and SPAC1782.03, they have been called SpSaf2 and SpSaf3, respectively. To confirm that they interacted with SpNTC components, they were tagged at their endogenous loci with the GFP, TAP, or HA3-TAP epitopes. Standard co-immunoprecipitations validated their interactions with SpDre4 and SpPrp19 (Figure S3). Furthermore, following TAP, 2D-LC mass spectrometric analysis revealed that SpSaf2 and SpSaf3 associate with many splicing factors (Figure 3 and Table S3). There were also many background proteins identified, typical of low abundance proteins (Table S3). Indeed SpSaf1, SpSaf2 and SpSaf3 are considerably less abundant proteins (43-, 55-, and 10-fold, respectively) than SpPrp17 as determined by quantitative immunoblotting (Figure 8A). This prevented the determination of SpSaf1, SpSaf2 or SpSaf3 TAP complex size by sucrose gradient sedimentation and clearly indicate that, like SpDre4, these proteins are not core NTC components.


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)

Characterization of S. pombe Safs.A) Protein G pull-downs from the indicated S. pombe strains were blotted with antibodies to the HA epitope. The bands with asterisks correspond to the indicated proteins and were quantified relative to background. B and C) Spores from the (B) saf2::ura4+/saf2+ and (D) saf3::ura4+/saf3+ diploids were germinated in minimal medium lacking uracil. Cells were fixed in formaldehyde at 15 and 40 h, respectively, and stained with DAPI. D and E) RNA was purified from wildtype cells grown at 32°C, prp2-1 cells grown at 25°C(−) or shifted to 36°C (+) for 4 hours, or from spores germinated at 32°C for 24 h from saf2::ura4+/saf2+ (D) and saf3::ura4+/saf3+ (E) diploids in medium lacking uracil. 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. Arrows indicate the position of prescursor and mature RNA species.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046128&req=5

pone-0016719-g008: Characterization of S. pombe Safs.A) Protein G pull-downs from the indicated S. pombe strains were blotted with antibodies to the HA epitope. The bands with asterisks correspond to the indicated proteins and were quantified relative to background. B and C) Spores from the (B) saf2::ura4+/saf2+ and (D) saf3::ura4+/saf3+ diploids were germinated in minimal medium lacking uracil. Cells were fixed in formaldehyde at 15 and 40 h, respectively, and stained with DAPI. D and E) RNA was purified from wildtype cells grown at 32°C, prp2-1 cells grown at 25°C(−) or shifted to 36°C (+) for 4 hours, or from spores germinated at 32°C for 24 h from saf2::ura4+/saf2+ (D) and saf3::ura4+/saf3+ (E) diploids in medium lacking uracil. 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. Arrows indicate the position of prescursor and mature RNA species.
Mentions: Two other predicted proteins were identified in the S. pombe NTC component TAPs discussed above with high sequence coverage (Figure 3 and Tables S2 and S3). Encoded by ORFs SPAC2F3.14c and SPAC1782.03, they have been called SpSaf2 and SpSaf3, respectively. To confirm that they interacted with SpNTC components, they were tagged at their endogenous loci with the GFP, TAP, or HA3-TAP epitopes. Standard co-immunoprecipitations validated their interactions with SpDre4 and SpPrp19 (Figure S3). Furthermore, following TAP, 2D-LC mass spectrometric analysis revealed that SpSaf2 and SpSaf3 associate with many splicing factors (Figure 3 and Table S3). There were also many background proteins identified, typical of low abundance proteins (Table S3). Indeed SpSaf1, SpSaf2 and SpSaf3 are considerably less abundant proteins (43-, 55-, and 10-fold, respectively) than SpPrp17 as determined by quantitative immunoblotting (Figure 8A). This prevented the determination of SpSaf1, SpSaf2 or SpSaf3 TAP complex size by sucrose gradient sedimentation and clearly indicate that, like SpDre4, these proteins are not core NTC components.

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