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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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Model of Prp19 organization in the S. pombe NTC.
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pone-0016719-g009: Model of Prp19 organization in the S. pombe NTC.

Mentions: Conserved from yeasts to human [59], [60], [61], Prp17 is considered to be a second step factor present only in activated spliceosomes (for example, [5]) although other evidence suggests it associates with snRNPs prior to the first step of splicing [48], [52]. Here, we establish that Prp17 binds directly to the Prp19 WD40 domain and is present in splicing complexes in a 1∶1 stoichiometry with Prp19. Based on the combined evidence, we conclude that Prp17 is a bona fide NTC component, constitutively associated with Prp19 (Figure 9). The Prp17 N-terminus, which interacts with Prp19, does not encode a recognizable motif but some temperature-sensitive prp17 mutations map to this region [62]. Given the Prp17 domain architecture, and numerous genetic interactions between prp17Δ and other splicing mutants [62], [63], it is likely that Prp17 stabilizes associations among NTC components or between the NTC and snRNPs. It will be interesting to determine if Prp17 associates with other splicing factors through its WD40 domains and if so, to learn their identities.


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)

Model of Prp19 organization in the S. pombe NTC.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016719-g009: Model of Prp19 organization in the S. pombe NTC.
Mentions: Conserved from yeasts to human [59], [60], [61], Prp17 is considered to be a second step factor present only in activated spliceosomes (for example, [5]) although other evidence suggests it associates with snRNPs prior to the first step of splicing [48], [52]. Here, we establish that Prp17 binds directly to the Prp19 WD40 domain and is present in splicing complexes in a 1∶1 stoichiometry with Prp19. Based on the combined evidence, we conclude that Prp17 is a bona fide NTC component, constitutively associated with Prp19 (Figure 9). The Prp17 N-terminus, which interacts with Prp19, does not encode a recognizable motif but some temperature-sensitive prp17 mutations map to this region [62]. Given the Prp17 domain architecture, and numerous genetic interactions between prp17Δ and other splicing mutants [62], [63], it is likely that Prp17 stabilizes associations among NTC components or between the NTC and snRNPs. It will be interesting to determine if Prp17 associates with other splicing factors through its WD40 domains and if so, to learn their identities.

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