FIGURE 4.

Detection of a U2/U6 di-snRNP, U4 snRNP, and free U6 RNA in the triple mutant strain. Analysis of wild-type and triple mutant (starred lanes) whole-cell extracts with (dotted lanes) or without incubation with ATP by nondenaturing 4% polyacrylamide gel electrophoresis. RNA was transferred to a membrane, which was then serially probed with ³²P-labeled DNA oligomer complementary to the RNA indicated above each panel. Bold labels indicate snRNPs and plain labels indicate snRNAs. Black boxes highlight stable U2/U6 RNA complexes present in the triple mutant strain. In lanes 11–12, extracts were deproteinized at low temperature by phenol/chloroform extraction, leaving only RNA. In lanes 13–14, DNA oligomer complementary to positions of 89–111 of U2 snRNA was added to promote RNase H cleavage of U2 (“cut U2”). U6* appears to be a truncated form of U6 RNA. Yeast U5 snRNA is produced as long (U5L) and short (U5S) species.