Guillaume Rigaut

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

The characterization of a novel yeast-splicing factor, Luc7p, is presented. The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC). Luc7p is similar in sequence to metazoan proteins that have arginine-serine and arginine-glutamic acid repeat sequences characteristic of a family of splicing factors. We show that Luc7p is a component of yeast U1 snRNP and is essential for vegetative growth. The composition of yeast U1 snRNP is altered in luc7 mutant strains. Extracts of these strains are unable to support any of the defined steps of splicing unless recombinant Luc7p is added. Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5' splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains. By use of reporters that have two competing 5' splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC. CBC can be coprecipitated with U1 snRNP from wild-type, but not from luc7, yeast strains. These data suggest that the loss of Luc7p disrupts U1 snRNP-CBC interaction, and that this interaction contributes to normal 5' splice site recognition.

Complex cellular functions involve large networks of interactions. Pre-mRNA splicing and transcription are thought to be coupled by the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). In yeast, the U1 snRNP subunit Prp40 was proposed to mediate cotranscriptional recruitment of early splicing factors through binding of its WW domains to the Pol II CTD. Here we investigate the role of Prp40 in splicing with an emphasis on the role of the WW domains, which might confer protein-protein interactions among the splicing and transcriptional machineries. Affinity purification revealed that Prp40 and Snu71 form a stable heterodimer that stably associates with the U1 snRNP only in the presence of Nam8, a known regulator of 5' splice site recognition. However, the Prp40 WW domains were dispensable for yeast viability. In their absence, no defect in splicing in vivo, U1 or U2 snRNP recruitment in vivo, or early splicing complex assembly in vitro was detected. We conclude that the WW domains of Prp40 do not mediate essential coupling between U1 snRNP and Pol II. Instead, delays in cotranscriptional U5 snRNP and Prp19 recruitment and altered spliceosome formation in vitro suggest that Prp40 WW domains assist in late steps of spliceosome assembly.