Nuclear pre-mRNA splicing proceeds via two mechanistically conserved consecutive trans-esterification reactions catalyzed by the spliceosome. The ordered coalescence of spliceosomal snRNPs and splicing factors on the pre-mRNA, coupled with essential spliceosomal rearrangements poise the splice-sites in proximity for the two catalytic reactions, ensuring intron removal and exon ligation to yield functional mRNA (reviewed in Will and Lührmann, 2006). Scope of the study The S. cerevisiae splicing factors Prp18 and Slu7 and their human homologs function during second catalytic reaction. In S. cerevisiae, Slu7 is essential, whereas Prp18 is dispensable at temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Frank et al., 1992; Horowitz and Abelson, 1993b; reviewed in Umen and Guthrie, 1995). Slu7 acts in concert with Prp18 and their direct interaction is required for their stable spliceosomal association (Zhang and Schwer, 1997; James et al., 2002). In vitro studies indicate that both the factors are dispensable for splicing of introns with short distances between branch nucleotide to 3’ splice-site (Brys and Schwer, 1996; Zhang and Schwer, 1997). Furthermore, mutational analyses of Slu7 and Prp18 have defined their functional domains/motifs (Frank and Guthrie, 1992; Bacíková and Horowitz, 2002; James et al., 2002). In this study, we have examined functions for the predicted homologs of Slu7 and Prp18 in fission yeast; an evolutionarily divergent organism where splicing mechanisms are not well understood and whose genome harbors genes with predominantly multiple introns with degenerate splice-junction sequences. Towards this goal, a combinatorial approach employing genetic and biochemical methods was undertaken to understand splicing functions and interactions of SpSlu7 and SpPrp18. Our mutational analysis of these protein factors provided an overview of the domains/motifs critical for their in vivo functions. Lastly our analysis of components of the budding yeast Cef1p-associated complex show novel interactions and splicing functions for two uncharacterized, yet evolutionarily conserved proteins. Conserved fission yeast splicing proteins SpSlu7 and SpPrp18 are essential for pre-mRNA splicing but have altered spliceosomal associations and functions Analyzing conserved splicing factors in evolutionarily divergent organisms is an important means to gain deeper functional insights on splicing mechanisms in genomes with varied gene architecture. We initiated our analysis of the ‘predicted’ S. pombe second-step splicing factors: spprp18+ and spslu7+, by genetically depleting these factors. We find spprp18+ is essential for viability, unlike budding yeast PRP18; while SLU7 is essential in both yeasts. The complete essentiality of both these fission yeast factors, prompted us to create conditional-lethal thiamine repressible ‘switch-off’ strains to probe their splicing functions. Through semi-quantitative RT-PCR and northern blot analysis we demonstrate splicing defects for tfIId+ pre-mRNAs upon metabolic depletion of spprp18+ or spslu7+, thus linking their essentiality to a role in pre-mRNA splicing. Further we examined whether their requirement as splicing factors is governed by specific intronic features. We find both factors are required in vivo for removal of several introns. However, for the introns tested, their functions are not strictly correlated with intron length, number, position or the branch-nucleotide to 3’ splice-site distance. The latter features dictate the need for their S. cerevisiae homologs. Strikingly the lack of either one of these essential proteins, arrests splicing before the first catalytic step; implicating possible functions early in spliceosome assembly even before any catalytic event, as opposed to budding yeast Slu7 and Prp18, which are second-step factors assembling late onto the spliceosome after the first splicing reaction. Given the different splicing arrest point, on depletion of SpSlu7 and SpPrp18, we investigated through yeast two-hybrid and co-immunoprecipitation assays whether the direct interaction between these proteins is conserved. We find despite being nuclear localized these proteins do not interact in either of the assays employed. A structural basis for the lack of interaction was provided by our homology modeling of SpPrp18, that was based on the crystal structure of S. cerevisiae Prp1879 (Jiang et al., 2000). Together these data raise the possibility of contextual functions and interactions for these conserved proteins that varies with changes in gene architecture. This likelihood is strengthened by our reciprocal genetic complementation tests; wherein we find that SpSlu7 and SpPrp18 cannot complement the corresponding S. cerevisiae null alleles and vice versa. Additionally, the human homologs, hSlu7 and hPrp18 also failed to rescue null alleles for spslu7+ and spprp18+. To understand the likely point of coalescence of SpSlu7 and SpPrp18 on assembling spliceosomes, we probed their snRNP associations through co-immunoprecipitation analysis. Our data revealed interaction of SpSlu7 with the U2, U5 and U6 snRNPs at moderate salt concentrations with the interaction with U5 snRNP being retained at higher salt conditions. SpPrp18, on the other hand, showed only a very weak association with U5 snRNP. Our analysis thus indicates that the assembly and step of action for “predicted” late-acting splicing factors in fission yeast differs from that in budding yeast, implicating novel interactions and functions for these fission yeast splicing factors. Mutational analysis of fission yeast SpPrp18 and SpSlu7 identifies functional domains To examine the protein domains/motifs critical for the functions of SpPrp18 and SpSlu7, we have performed a mutational study. This analysis was important after our findings that these factors are early acting and do not interact. The data gathered would shed light on the contribution of different domains/motifs in the functional diversification of these factors. Guided by the findings of Bacíková and Horowitz (2002); site-specific missense mutants were created in the highly conserved carboxyl-terminus (CR domain and helix 5) of SpPrp18. Additionally, site-specific missense mutants were generated in a conserved amino-terminus domain that is absent in budding yeast Prp18. Our data showed mutants in the highly conserved helix 5 and the CR domain of SpPrp18 to be recessive and non-functional, despite being stably expressed. This contrasts with the temperature-sensitivity conferred by similar mutants in homologous residues in budding yeast Prp18 (Bacíková and Horowitz, 2002). We speculate that the essentiality of the CR domain and helix 5 mutants of SpPrp18 arises due to a defect in spliceosomal association. However, the mutants in conserved residues in the protein’s amino-terminal domain are phenotypically wild type at various growth temperatures tested, suggesting redundant functions for these residues. Our data, based on analysis of a single missense mutant in the highly conserved zinc knuckle motif of SpSlu7, ascribes essential functions for the zinc knuckle motif. We find the mutant to be recessive and non-functional despite stable expression and normal cellular localization of the mutant protein. This contrasts with the behavior of zinc knuckle mutants in budding yeast and human Slu7. Budding yeast Slu7 mutants are functionally wild type and human Slu7 mutants have an altered cellular localization (Frank and Guthrie, 1992; James et al., 2002; Shomron et al., 2004). Possible roles for the zinc knuckle motif of SpSlu7 could be in facilitating interaction of SpSlu7 with U5 snRNA or even with some protein factor. Functional analysis of budding yeast Cef1p-associated complex SpSlu7 and its budding yeast homolog ScSlu7 co-purify with Cdc5/Cef1 in a complex of ~30 proteins together with U2, U5 and U6 snRNAs (Gavin et al., 2002; Ohi et al., 2002). Functional characterization of six proteins of the budding yeast Cef1p complex: Ydl209c (Cwc2/Ntc40), Ycr063w (Cwc14/Bud31), Yju2 (Cwc16), Ygr278w (Cwc22), Ylr424w (Spp382/Ntr1) and Ygl128c (Cwc23) was initiated using a combination of genetic and biochemical approaches. We probed direct protein-protein interactions between members of the Cef1p-associated complex by yeast two-hybrid assays. We also examined the pre-mRNA splicing roles for an essential factor, Yju2/Cwc16 and for a non-essential factor, Ycr063w/Cwc14. Our data reveals direct interaction between Yju2 and early acting factors, Syf1/Ntc90 and Clf1/Ntc77. Similarly interaction of Ydl209c/Cwc2 with early acting splicing factors, Prp19, Syf1/Ntc90 and Clf1/Ntc77 was noted. We created a temperature-sensitive expression strain for YJU2 using a temperature-sensitive Gal4 transcription trans-activator (Chakshusmathi et al., 2004; Mondal et al., 2007) to interrogate the splicing functions of YJU2. RT-PCR and northern blot assays show that depletion of YJU2 causes splicing defects for intron containing pre-mRNAs. We predict early splicing functions for YJU2 as is known for its interacting partners. Furthermore, we find that genetic depletion of the non-essential factor YCR063w causes temperature-sensitivity as has been reported for a few other factors (for e.g. Prp17, Lea1, Snt309/Ntc25, Ecm2) of Cef1p-associated complex (Jones et al, 1995; Chen et al., 1998). Although our yeast two-hybrid data does not reveal any direct interactions between Ycr063w and other proteins of the Cef1p-associated complex, we probed its functions through in vitro splicing assays. Splicing extracts from ycr063w/ycr063w cells show compromised second-step splicing at higher temperatures, thereby implying an auxiliary function for Ycr063w in stabilizing some functionally critical interactions during splicing. These studies employing complementary genetic and biochemical approaches implicate functional divergence of conserved predicted ‘second-step’ fission yeast factors, SpSlu7 and SpPrp18, suggesting co-evolution of splicing factors with changes in genome architecture and intron-exon structure. Our studies on Cef1p-associated complex show novel interactions and implicate pre-mRNA splicing functions for two previously uncharacterized proteins.