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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-ScUrn1/SpDre4 interaction.A) S. cerevisiae strain pJ69-4A was cotransformed with bait plasmids expressing the indicated regions of ScURN1. + indicate growth and – denotes no growth on selective media. ß-galactosidase activity (represented by relative light units) of the transformants is plotted in the right panels. (B–E) Continuous size distribution analysis of sedimentation velocity data of ScPrp19, His6-ScUrn1, and ScPrp19:His6-ScUrn1. Calculated c(s) is plotted versus sedimentation coefficients (s) for (B) ScPrp19, (C) His6-ScUrn1, (D) ScPrp19:His6-ScUrn1 in a 1∶1 molar ratio and (E) ScPrp19:His6-ScUrn1 in a 1∶2.5 molar ratio. Each s peak is labeled with predicted molecular mass (kDa). ScPrp19 concentrations were constant, 10 mM, with His6-ScUrn1 concentrations varied to the indicated molar ratio. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm.
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pone-0016719-g004: Characterization of Prp19-ScUrn1/SpDre4 interaction.A) S. cerevisiae strain pJ69-4A was cotransformed with bait plasmids expressing the indicated regions of ScURN1. + indicate growth and – denotes no growth on selective media. ß-galactosidase activity (represented by relative light units) of the transformants is plotted in the right panels. (B–E) Continuous size distribution analysis of sedimentation velocity data of ScPrp19, His6-ScUrn1, and ScPrp19:His6-ScUrn1. Calculated c(s) is plotted versus sedimentation coefficients (s) for (B) ScPrp19, (C) His6-ScUrn1, (D) ScPrp19:His6-ScUrn1 in a 1∶1 molar ratio and (E) ScPrp19:His6-ScUrn1 in a 1∶2.5 molar ratio. Each s peak is labeled with predicted molecular mass (kDa). ScPrp19 concentrations were constant, 10 mM, with His6-ScUrn1 concentrations varied to the indicated molar ratio. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm.

Mentions: To define the physical association of ScUrn1 with ScPrp19, we first refined the interaction domains by directed two-hybrid analysis. ScUrn1 sequences including the FF domain, the structure of which has been determined [55], were sufficient for Prp19 interaction (Figure 4A). The Prp19-interacting domain was fused to MBP, produced in E. coli, and purified. MBP-ScUrn1(165–274), but not MBP, was able to specifically bind Ni2+ beads coated with His6-ScPrp19(144–503) (Figure 2D).


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-ScUrn1/SpDre4 interaction.A) S. cerevisiae strain pJ69-4A was cotransformed with bait plasmids expressing the indicated regions of ScURN1. + indicate growth and – denotes no growth on selective media. ß-galactosidase activity (represented by relative light units) of the transformants is plotted in the right panels. (B–E) Continuous size distribution analysis of sedimentation velocity data of ScPrp19, His6-ScUrn1, and ScPrp19:His6-ScUrn1. Calculated c(s) is plotted versus sedimentation coefficients (s) for (B) ScPrp19, (C) His6-ScUrn1, (D) ScPrp19:His6-ScUrn1 in a 1∶1 molar ratio and (E) ScPrp19:His6-ScUrn1 in a 1∶2.5 molar ratio. Each s peak is labeled with predicted molecular mass (kDa). ScPrp19 concentrations were constant, 10 mM, with His6-ScUrn1 concentrations varied to the indicated molar ratio. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016719-g004: Characterization of Prp19-ScUrn1/SpDre4 interaction.A) S. cerevisiae strain pJ69-4A was cotransformed with bait plasmids expressing the indicated regions of ScURN1. + indicate growth and – denotes no growth on selective media. ß-galactosidase activity (represented by relative light units) of the transformants is plotted in the right panels. (B–E) Continuous size distribution analysis of sedimentation velocity data of ScPrp19, His6-ScUrn1, and ScPrp19:His6-ScUrn1. Calculated c(s) is plotted versus sedimentation coefficients (s) for (B) ScPrp19, (C) His6-ScUrn1, (D) ScPrp19:His6-ScUrn1 in a 1∶1 molar ratio and (E) ScPrp19:His6-ScUrn1 in a 1∶2.5 molar ratio. Each s peak is labeled with predicted molecular mass (kDa). ScPrp19 concentrations were constant, 10 mM, with His6-ScUrn1 concentrations varied to the indicated molar ratio. AU experiments were conducted at 22°C at a speed of 30,000 rpm and concentration profiles measured at 280 nm.
Mentions: To define the physical association of ScUrn1 with ScPrp19, we first refined the interaction domains by directed two-hybrid analysis. ScUrn1 sequences including the FF domain, the structure of which has been determined [55], were sufficient for Prp19 interaction (Figure 4A). The Prp19-interacting domain was fused to MBP, produced in E. coli, and purified. MBP-ScUrn1(165–274), but not MBP, was able to specifically bind Ni2+ beads coated with His6-ScPrp19(144–503) (Figure 2D).

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