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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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Characterization of Prp19-Prp17 interaction.A) A fraction of the SpPrp17-TAP eluate was analyzed by silver staining. Positions of markers are indicated. B) SpPrp17-HA3-TAP eluate was resolved on a 10 to 30% sucrose gradient, and fractions were collected from the bottom (fraction1). These were resolved by SDS-PAGE and immunoblotted with anti-HA to detect the migration of Prp17. Migration of of catalase (11.3S) and thyroglobulin (19S) collected from parallel gradients is indicated with asterisks. C) Four representative class averages of SpPrp17-TAP particles in negative stain. The number of particles in each projection average is shown in the lower right corner of each average. Side length of individual panels is 537.6 Å. D) Purified and soluble MBP, MBP-ScUrn(165–274), or MBP-ScPrp17 (Inputs) were incubated with Ni-NTA beads alone or Ni-NTA beads coated with His6-ScPrp19(144–503). Proteins bound to the beads after washing were detected by Coomassie blue staining. Asterisks indicate MBP-ScPrp17 and MBP-ScUrn1 fragments pulled down by the ScPrp19 WD40 domain. The Ni-NTA beads alone did not pull down MBP or MBP fusion proteins, but did pull down some non-specifically binding bacterial proteins. E) An anti-HA immunoprecipitate from S. pombe cwf7-HA prp19-Myc13 prp17-Myc13 cells was blotted for the presence of Myc-tagged proteins. Bands were quantified on an Odyssey instrument. F) Coomassie stained gel of purified MBP-ScPrp17 produced in E. coli. Note the degradation bands. G) Continuous size distribution analysis of sedimentation velocity data of MBP-ScPrp17. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm. H) SpPrp19-TAP complex was isolated from a S. pombe prp19-TAP cwf2-GFP prp17-Myc13 strain and a portion of the eluate was probed for the presence of SpPrp17 and SpCwf2. The remainder of the eluate was 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-g002: Characterization of Prp19-Prp17 interaction.A) A fraction of the SpPrp17-TAP eluate was analyzed by silver staining. Positions of markers are indicated. B) SpPrp17-HA3-TAP eluate was resolved on a 10 to 30% sucrose gradient, and fractions were collected from the bottom (fraction1). These were resolved by SDS-PAGE and immunoblotted with anti-HA to detect the migration of Prp17. Migration of of catalase (11.3S) and thyroglobulin (19S) collected from parallel gradients is indicated with asterisks. C) Four representative class averages of SpPrp17-TAP particles in negative stain. The number of particles in each projection average is shown in the lower right corner of each average. Side length of individual panels is 537.6 Å. D) Purified and soluble MBP, MBP-ScUrn(165–274), or MBP-ScPrp17 (Inputs) were incubated with Ni-NTA beads alone or Ni-NTA beads coated with His6-ScPrp19(144–503). Proteins bound to the beads after washing were detected by Coomassie blue staining. Asterisks indicate MBP-ScPrp17 and MBP-ScUrn1 fragments pulled down by the ScPrp19 WD40 domain. The Ni-NTA beads alone did not pull down MBP or MBP fusion proteins, but did pull down some non-specifically binding bacterial proteins. E) An anti-HA immunoprecipitate from S. pombe cwf7-HA prp19-Myc13 prp17-Myc13 cells was blotted for the presence of Myc-tagged proteins. Bands were quantified on an Odyssey instrument. F) Coomassie stained gel of purified MBP-ScPrp17 produced in E. coli. Note the degradation bands. G) Continuous size distribution analysis of sedimentation velocity data of MBP-ScPrp17. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm. H) SpPrp19-TAP complex was isolated from a S. pombe prp19-TAP cwf2-GFP prp17-Myc13 strain and a portion of the eluate was probed for the presence of SpPrp17 and SpCwf2. The remainder of the eluate was 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: Prp17 has been identified in isolations of the splicing apparatus from multiple organisms [1] including yeasts [24], [52], and ScPrp17 co-purifies the U2, U5 and U6 snRNAs [48]. To determine the SpPrp17 associated splicing factors, it was tagged with the TAP or HA3-TAP epitope and purified. Proteins present in SpPrp17-TAP complexes were visualized by silver staining (Figure 2A) and identified by 2D-LC-MS/MS (Figure 3 and Table S2). The compilation of associated splicing factors was compared to that of other S. pombe NTC components such as SpCdc5 [24], SpCwf2 and SpPrp19 (Figure 2A, Figure 3, and Table S2), which all co-purified primarily U2, U5 and NTC components. The SpPrp17-HA3-TAP eluate sedimented on a sucrose gradient with a single peak of comparable size to the SpCdc5-TAP complex (Figure 2B) [24], [53] indicating that, like Cdc5 [53], all Prp17 is associated with this complex. Furthermore, the complex purified by SpPrp17-TAP appears by electron microscopy to be very similar in size and homogeneity (Figure 2C and data not shown) to that purified by Cdc5 [54]. We conclude that SpPrp17 is a stable component of the NTC.


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 Prp19-Prp17 interaction.A) A fraction of the SpPrp17-TAP eluate was analyzed by silver staining. Positions of markers are indicated. B) SpPrp17-HA3-TAP eluate was resolved on a 10 to 30% sucrose gradient, and fractions were collected from the bottom (fraction1). These were resolved by SDS-PAGE and immunoblotted with anti-HA to detect the migration of Prp17. Migration of of catalase (11.3S) and thyroglobulin (19S) collected from parallel gradients is indicated with asterisks. C) Four representative class averages of SpPrp17-TAP particles in negative stain. The number of particles in each projection average is shown in the lower right corner of each average. Side length of individual panels is 537.6 Å. D) Purified and soluble MBP, MBP-ScUrn(165–274), or MBP-ScPrp17 (Inputs) were incubated with Ni-NTA beads alone or Ni-NTA beads coated with His6-ScPrp19(144–503). Proteins bound to the beads after washing were detected by Coomassie blue staining. Asterisks indicate MBP-ScPrp17 and MBP-ScUrn1 fragments pulled down by the ScPrp19 WD40 domain. The Ni-NTA beads alone did not pull down MBP or MBP fusion proteins, but did pull down some non-specifically binding bacterial proteins. E) An anti-HA immunoprecipitate from S. pombe cwf7-HA prp19-Myc13 prp17-Myc13 cells was blotted for the presence of Myc-tagged proteins. Bands were quantified on an Odyssey instrument. F) Coomassie stained gel of purified MBP-ScPrp17 produced in E. coli. Note the degradation bands. G) Continuous size distribution analysis of sedimentation velocity data of MBP-ScPrp17. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm. H) SpPrp19-TAP complex was isolated from a S. pombe prp19-TAP cwf2-GFP prp17-Myc13 strain and a portion of the eluate was probed for the presence of SpPrp17 and SpCwf2. The remainder of the eluate was 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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getmorefigures.php?uid=PMC3046128&req=5

pone-0016719-g002: Characterization of Prp19-Prp17 interaction.A) A fraction of the SpPrp17-TAP eluate was analyzed by silver staining. Positions of markers are indicated. B) SpPrp17-HA3-TAP eluate was resolved on a 10 to 30% sucrose gradient, and fractions were collected from the bottom (fraction1). These were resolved by SDS-PAGE and immunoblotted with anti-HA to detect the migration of Prp17. Migration of of catalase (11.3S) and thyroglobulin (19S) collected from parallel gradients is indicated with asterisks. C) Four representative class averages of SpPrp17-TAP particles in negative stain. The number of particles in each projection average is shown in the lower right corner of each average. Side length of individual panels is 537.6 Å. D) Purified and soluble MBP, MBP-ScUrn(165–274), or MBP-ScPrp17 (Inputs) were incubated with Ni-NTA beads alone or Ni-NTA beads coated with His6-ScPrp19(144–503). Proteins bound to the beads after washing were detected by Coomassie blue staining. Asterisks indicate MBP-ScPrp17 and MBP-ScUrn1 fragments pulled down by the ScPrp19 WD40 domain. The Ni-NTA beads alone did not pull down MBP or MBP fusion proteins, but did pull down some non-specifically binding bacterial proteins. E) An anti-HA immunoprecipitate from S. pombe cwf7-HA prp19-Myc13 prp17-Myc13 cells was blotted for the presence of Myc-tagged proteins. Bands were quantified on an Odyssey instrument. F) Coomassie stained gel of purified MBP-ScPrp17 produced in E. coli. Note the degradation bands. G) Continuous size distribution analysis of sedimentation velocity data of MBP-ScPrp17. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm. H) SpPrp19-TAP complex was isolated from a S. pombe prp19-TAP cwf2-GFP prp17-Myc13 strain and a portion of the eluate was probed for the presence of SpPrp17 and SpCwf2. The remainder of the eluate was 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: Prp17 has been identified in isolations of the splicing apparatus from multiple organisms [1] including yeasts [24], [52], and ScPrp17 co-purifies the U2, U5 and U6 snRNAs [48]. To determine the SpPrp17 associated splicing factors, it was tagged with the TAP or HA3-TAP epitope and purified. Proteins present in SpPrp17-TAP complexes were visualized by silver staining (Figure 2A) and identified by 2D-LC-MS/MS (Figure 3 and Table S2). The compilation of associated splicing factors was compared to that of other S. pombe NTC components such as SpCdc5 [24], SpCwf2 and SpPrp19 (Figure 2A, Figure 3, and Table S2), which all co-purified primarily U2, U5 and NTC components. The SpPrp17-HA3-TAP eluate sedimented on a sucrose gradient with a single peak of comparable size to the SpCdc5-TAP complex (Figure 2B) [24], [53] indicating that, like Cdc5 [53], all Prp17 is associated with this complex. Furthermore, the complex purified by SpPrp17-TAP appears by electron microscopy to be very similar in size and homogeneity (Figure 2C and data not shown) to that purified by Cdc5 [54]. We conclude that SpPrp17 is a stable component of the NTC.

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
Related in: MedlinePlus