Figure 6.

Stabilizing the telestem can interfere with yeast growth but does not prevent U4/U6 formation in vivo. (A) The effects of telestem mutations were assessed by 10-fold serial dilution and plating of yeast containing the indicated U6 alleles onto YPD/agar media followed by incubation of the plates at the indicated temperatures. The U6_{BASE + ΔBULGE} mutation was unable to survive the 5-FOA selection and thus is recessive lethal. (B) Solution hybridization assay of whole-cell RNA. RNA was extracted from the indicated strains at 4°C to preserve U4/U6 base-pairing, hybridized to [³²P]-labeled DNA oligonucleotides complementary to U1, U4 or U6 at 37°C, and analyzed by non-denaturing 9% PAGE followed by phosphorimaging. U6-SH probe was used in lanes 2, 6, 8 and 10 and U6-SH-MUT probe was used in lanes 4 and 12 to accommodate the A34G/A35G mutations. U4/U6 exhibits different mobilities when hybridized to the U4 or U6 probes. Multiple U6 bands indicative of structural heterogeneity were observed in some of the mutant strains. (C) Analysis of the fraction of U4 present in each strain as a U4/U6 complex. Also shown are the relative total amounts of U4 and U6, expressed as ratios to U1, normalized to the U6_WT strain. Error bars represent SD of data obtained from three replicate experiments.