U6 snRNA made by RNAP II is unstable in vivo. (A) Growth curve of an SNR6 disruption strain containing pRS314-SNR6 (WT) or pRS314-GAL-SNR6-II after replacement of YEP-galactose medium with YEP-glucose at time 0 to repress transcription of the GAL-SNR6-II allele. Both strains were diluted to keep the optical density at 600 nm (OD600) less than 1. The “effective OD600” value is corrected for this dilution. (B) Total RNA extracted from cells collected at the indicated times was subjected to primer extension to detect U1, U4, and U6 snRNAs and U3 snoRNA, which is transcribed from two loci (A,B) as an intron-containing precursor (pre-U3). (C) Quantitation of U6, U4, and mature U3 RNA present in the gel from panel B relative to the WT strain immediately before shift from galactose to glucose (0 h). Values are normalized to U1 snRNA in each sample to control for variable RNA recovery. (D) Quantitation of pre-U3 snoRNA present in the gel from panel B relative to the WT strain immediately before shift from galactose to glucose (0 h). Values are normalized as in panel C.
