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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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Mass spectrometric analysis of S. cerevisiae splicing associated factors.Proteins are categorized by sub-complex with the number of spectral counts and percent sequence coverage provided. Components present at less than 5% sequence coverage or with less than five distinct peptides were not included in the compilation of splicing factors based on subcomplexes. Full analyses of mass spectrometric data are provided in Tables S4 and S5. UNK = unknown.
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pone-0016719-g006: Mass spectrometric analysis of S. cerevisiae splicing associated factors.Proteins are categorized by sub-complex with the number of spectral counts and percent sequence coverage provided. Components present at less than 5% sequence coverage or with less than five distinct peptides were not included in the compilation of splicing factors based on subcomplexes. Full analyses of mass spectrometric data are provided in Tables S4 and S5. UNK = unknown.

Mentions: Sequences encoding a TAP or HA3-TAP epitope were added to the 3′end of the dre4+ open reading frame to enable SpDre4 interacting proteins to be purified and identified by mass spectrometry (Figure 3 and Table S3). SpDre4-TAP co-purified a large number of splicing factors in addition to SpPrp19 including components of the U2 and U5 snRNPs and the NTC (Figure 3). Proteins known to be involved in other cellular processes were not identified to any significant extent although there was significant background typical of low abundance proteins (Table S3). Indeed, the SpDre4-TAP complex was not abundant enough to be visualized following sucrose gradient sedimentation (data not shown). Further evidence that SpDre4-TAP associates with splicing complexes was the presence of U2, U5 and U6 snRNAs in the TAP eluate, as was found in the SpPrp17-TAP, SpPrp19-TAP, and SpCwf2-TAP eluates (Figure 5B). These associations have been conserved throughout evolution as a ScUrn1-TAP eluate, contained a similar set of U2, U5 and NTC splicing factors as determined by 2D-LC-mass spectrometry (Figure 6 and Tables S4 and S5).


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)

Mass spectrometric analysis of S. cerevisiae splicing associated factors.Proteins are categorized by sub-complex with the number of spectral counts and percent sequence coverage provided. Components present at less than 5% sequence coverage or with less than five distinct peptides were not included in the compilation of splicing factors based on subcomplexes. Full analyses of mass spectrometric data are provided in Tables S4 and S5. UNK = unknown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046128&req=5

pone-0016719-g006: Mass spectrometric analysis of S. cerevisiae splicing associated factors.Proteins are categorized by sub-complex with the number of spectral counts and percent sequence coverage provided. Components present at less than 5% sequence coverage or with less than five distinct peptides were not included in the compilation of splicing factors based on subcomplexes. Full analyses of mass spectrometric data are provided in Tables S4 and S5. UNK = unknown.
Mentions: Sequences encoding a TAP or HA3-TAP epitope were added to the 3′end of the dre4+ open reading frame to enable SpDre4 interacting proteins to be purified and identified by mass spectrometry (Figure 3 and Table S3). SpDre4-TAP co-purified a large number of splicing factors in addition to SpPrp19 including components of the U2 and U5 snRNPs and the NTC (Figure 3). Proteins known to be involved in other cellular processes were not identified to any significant extent although there was significant background typical of low abundance proteins (Table S3). Indeed, the SpDre4-TAP complex was not abundant enough to be visualized following sucrose gradient sedimentation (data not shown). Further evidence that SpDre4-TAP associates with splicing complexes was the presence of U2, U5 and U6 snRNAs in the TAP eluate, as was found in the SpPrp17-TAP, SpPrp19-TAP, and SpCwf2-TAP eluates (Figure 5B). These associations have been conserved throughout evolution as a ScUrn1-TAP eluate, contained a similar set of U2, U5 and NTC splicing factors as determined by 2D-LC-mass spectrometry (Figure 6 and Tables S4 and S5).

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