CWC21 interacts genetically with genes encoding splicing factors. (A) Using SGA technology in a high-density E-MAP (epistatic miniarray profile) format, a strain harboring a deletion of CWC21(cwc21-Δ) was crossed to a panel of >500 mutants involved in various aspects of RNA processing (Wilmes et al. 2008). Growth rates of the double mutants were assessed by automated image analysis of colony size and interaction scores were derived as previously described (Schuldiner et al. 2005; Collins et al. 2006). Positive (e.g., suppression) and negative (e.g., synthetic lethality) genetic interactions are displayed above and below the zero baseline, respectively. (B) Scatterplot of genetic interactions observed for cwc21 (x-axis) and isy1 (y-axis) obtained from the RNA processing E-MAP.

Widespread defects in pre-mRNA splicing in double mutant yeast strains lacking Cwc21. cDNA prepared from RNA purified from wild-type (WT) and single and double gene deletion (mutant) strains was labeled with Cy3 and Cy5 dyes and hybridized to S. cerevisiae genome-wide Affymetrix tiling microarrays. Normalized probe intensity signals were determined for all intron-containing transcripts, excluding tRNAs and dubious ORFs. After normalization of expression levels between the intron-containing genes using averages of all probe signals for exon sequences, the average log₂ ratios of the mutant over wild-type expression differences were plotted in a fixed number of bins. (A) Plots of the average log₂ ratios relative to WT for all intron-containing genes in the cwc21-Δ and isy1-Δ single and double mutant stains. (B) Plots of the average log₂ ratios relative to WT for all intron-containing genes in the cwc21-Δ and bud13-Δ single and double mutant strains. Error bars in both plots indicate 95% confidence intervals. (C) Representative RT-PCR assay of spliced and unspliced transcripts from the GFI15 gene in the mutant strains analyzed by tiling microarrays. (D) Hierarchical clustering of the average expression level differences in intron sequences between mutants and WT strains, corrected for the mean signal across exons. Clusters are annotated for significantly enriched GO terms, with P-values calculated relative to a background set containing all genes (Pg) or intron- containing genes only (Pi). Additional clustering data and a list of genes in each cluster are provided in Supplemental Table 1.

Association of Cwc21 with Prp19 (NTC) components and spliceosomal snRNAs. (A) The mass-spectrometry data shown in Table 1 were merged with data from two large-scale proteomics studies of tag-affinity-purified S. cerevisiae protein complexes (Gavin et al. 2006; Krogan et al. 2006; Collins et al. 2007) and hierarchical clustering of the merged data sets was performed. Of >2000 proteins, Cwc21 clusters most closely with components of the NTC complex. The clustergram shown represents a small portion of the entire set of clustered data (data not shown). (B) Microarray profiling of RNA recovered from tag affinity-purified Cwc21 and Prp8 complexes. RNA recovered following each affinity purification was directly labeled with Cy3 and Cy5 dyes and hybridized to a custom tiling microarray (Hiley et al. 2005) that detects non-coding RNAs. Signals were sorted from highest to lowest signal for each snRNA. The scale indicates log₂ of the ratio of probe intensity in the pull-down to total RNA.