Sad1 is a core splicing factor necessary for spliceosome assembly. (A) Domain topology for S. cerevisiae Sad1, the human homolog of Sad1 (USP39), and a canonical active USP domain, with the catalytic residues denoted by asterisks. The ZnF-UBP domain is colored pale orange, a short linker domain is pale green, and the iUSP domain is colored slate blue. USP39 possesses an additional N-terminal RS domain. (B) The yeast pre-mRNA splicing microarray is composed of probes that detect the pre-mRNA intron (P), the exon (T) for total, and the exon/exon junction for the mature mRNA (M). SAD1 and sad1-1 cultures were grown at 25°C to mid-log phase and were then shifted for 30 min to 37°C. Total RNA was extracted and Cy3- and Cy5-labeled cDNA produced. The cDNAs were competitively hybridized to the array, and the resulting log2-ratios for each gene were plotted after performing centroid-linkage hierarchical clustering. Shown side by side are the heat maps of plots produced by two biological replicate experiments, a and b, where increasing amounts of yellow signal for the intron probe (P) reflects intron accumulation and is indicative of a pre-mRNA splicing defect. (C) sad1-1 splicing extracts were inactivated for 10 min at 37°C, and where indicated, a 10-fold decreasing dilution series of 105 nM full-length Sad1 was added. Pre-mRNA splicing reactions were incubated at 25°C in the presence of 2 mM ATP with Cy5-labeled pre-ACT1 for 30 min and then resolved on a 6% denaturing polyacrylamide gel. (D) Splicing extracts from sad1-1 were prepared and inactivated in a manner identical to that of the in vitro splicing assay in C, but the products of the reaction were resolved by native gel electrophoresis. An RNase H–catalyzed knockdown of the U6 snRNA in a SAD1 wild-type strain is shown for comparison.
