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 
). Spliceosome assembly occurs in discrete stages. The spliceosome assembly reaction is initiated when the 5′ and 3′ splice sites are recognized by the U1 and U2 snRNPs, respectively, forming complex A. The subsequent engagement of the U5.U4/U6 tri-snRNP to form complex B disrupts U1 binding to the pre-mRNA and triggers unwinding of the U4/U6 snRNA duplex, which is replaced by a U2/U6 snRNA duplex. 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. 5′splice site cleavage and lariat formation then occur in complex C, and finally the 3′ splice site is cleaved, the exons are ligated, and the spliceosome is released from the mRNA product.
Regulation of the structural rearrangements among snRNPs, the NTC, and other proteins is not fully understood but the transition from an inactive to an active spliceosome correlates with stable NTC binding 
. The NTC promotes new interactions between the U5 and U6 snRNAs with the pre-mRNA, and destabilizes interactions between the U6 snRNA and Sm-like (Lsm) proteins during complex C formation 
. However, the mechanistic details of the NTC's effects remain unknown.
In Saccharomyces cerevisiae
, the NTC has been purified as a distinct unit composed of about 10 proteins 
, many of which have been identified and are conserved in Schizosaccharomyces pombe
and human spliceosomal complexes 
. The namesake of the NTC, Prp19 (also known as S. cerevisiae
Pso4, human SNEV or NMP200, and Cwf8 in S. pombe
; hereafter referred to as Prp19 for orthologs in any organism), is a tetrameric protein that oligomerizes through a central coiled-coil domain in an anti-parallel manner 
(see ). Cef1 and Snt309 bind directly to the tetrameric coiled-coil domain 
. From each end of the tetramerization domain protrudes a dimer of the Prp19 N-terminal U-box domain 
, which confers E3 ubiquitin ligase activity to the protein 
. Also protruding from each end of Prp19's central stalk are two globular C-terminal WD40 domains. Given that WD40 repeats mediate protein-protein interactions, it is likely that each WD40 repeat interacts with other spliceosome components. However, only one NTC binding partner, Cwc2, has been identified for this domain 
Identification of Prp19 interactors.
Although first identified in S. cerevisiae
based on its role in pre-mRNA splicing, Prp19 has been implicated in other processes including DNA repair 
, recombination 
, sporulation 
, nuclear matrix structure, 
, and siRNA-mediated centromeric transcriptional silencing 
. Also, NTC components associate with activation-induced deaminase 
. Presently, it is unclear whether all these reported activities reflect splicing dependent or independent functions and whether Prp19 might be a multi-functional protein that interacts with distinct groups of proteins to carry out different functions. Certainly, the modular nature of its architecture might allow it to interact with both splicing and non-splicing factors.
In an attempt to identify the full complement of proteins capable of interacting with the WD40 domain of Prp19, we performed a global yeast two-hybrid screen using the S. cerevisiae
Prp19 WD40 domain as bait and went on to investigate whether positives in the screen directly interacted with this domain. In addition to its known interaction with Cwc2 
, we found that Prp19 binds directly to the splicing factor, Prp17, and the uncharacterized protein, Urn1. Interactions among NTC components are conserved between S. pombe
and S. cerevisiae 
and we used both yeast species here to examine biochemical properties, genetic interactions, and functions involving Prp17 and Urn1. For clarity, we will frequently refer to S. cerevisiae
proteins with the prefix Sc
and S. pombe
proteins with the prefix Sp
. From both yeasts, Sc
Dre4 purifications contain multiple known splicing factors, U2, U5, and U6 snRNAs and Sp
Dre4 is conditionally required for pre-mRNA splicing. Additionally, Sp
Dre4 co-purified four previously uncharacterized proteins essential for, or impact, pre-mRNA splicing, two of which are apparently absent in S. cerevisiae
. Thus, our combinatorial approaches led to the discovery of new splicing factors connected to the NTC and highlight a major function of the Prp19 WD40 domains as a scaffold for splicing proteins.