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.
Highlights
The spliceosome is a dynamic ribonucleoprotein complex that catalyzes the removal of introns from pre-mRNA in two discrete steps
Further reorganization occurs upon release of the U1 and U4 snRNPs and addition of the Prp19-associated NineTeen Complex (NTC) to form complex B*, marking spliceosomal activation. 59splice site cleavage and lariat formation occur in complex C, and the 39 splice site is cleaved, the exons are ligated, and the spliceosome is released from the mRNA product
Identification of Prp19 WD40 binding partners To identify binding partners of the atypical Prp19 WD40 domain [16], we performed a genome-wide two-hybrid screen in duplicate using as bait amino acids 146–503 of S. cerevisiae Prp19 fused to the Gal4 DNA-binding domain (Figure 1B) and an array of,6000 yeast strains expressing each S. cerevisiae ORF fused to the Gal4 activation domain [25,26]
Summary
The spliceosome is a dynamic ribonucleoprotein complex that catalyzes the removal of introns from pre-mRNA in two discrete steps. It is comprised of five snRNAs (U1, U2, U4, U5, and U6) bound both to intimately associated proteins that form ribonucleoprotein particles (snRNPs) and a host of other conserved protein factors, many whose function are not well understood (reviewed in [1]). The spliceosome assembly reaction is initiated when the 59 and 39 splice sites are recognized by the U1 and U2 snRNPs, respectively, forming complex A. Further reorganization occurs upon release of the U1 and U4 snRNPs and addition of the Prp19-associated NineTeen Complex (NTC) to form complex B*, marking spliceosomal activation. Further reorganization occurs upon release of the U1 and U4 snRNPs and addition of the Prp19-associated NineTeen Complex (NTC) to form complex B*, marking spliceosomal activation. 59splice site cleavage and lariat formation occur in complex C, and the 39 splice site is cleaved, the exons are ligated, and the spliceosome is released from the mRNA product
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