Expression of functional yeast U6 snRNA by RNAP II. (A) Diagram of construction of the GAL-SNR6-II allele. The wild type U6 (SNR6, orange) and U4 (SNR14, blue) snRNA genes are shown at top. Red lines span the mature RNA-coding regions. The SNR14 Nrd1–Nab3–Sen1 (NNS) terminator is downstream from the Rnt1 cleavage site (R). The GAL1 upstream activating sequence (UAS) is shown in green. Selected promoter and terminator elements are indicated by thick vertical lines. Numbers are base pairs upstream (−) or downstream (+) of the transcription start sites (bent arrow). (B) Northern blot of total cellular RNA from an SNR6 disruption strain containing a low-copy plasmid with WT SNR6 or a high-copy plasmid with the SNR6-II or SNR14-6-14 allele, using a U6 probe. (pre) presumptive primary transcript, (RCP) presumptive Rnt1 cleavage product, (U6-II) U6 snRNA synthesized by RNAP II. (C) Growth of an SNR6 disruption strain containing the indicated SNR6 allele on a low-copy plasmid on YEP medium containing glucose (gluc), raffinose (raff), or galactose (gal). (D) Primer extension analysis of U1 and U6 snRNAs in a U6 disruption strain containing the indicated SNR6 allele(s) on low-copy plasmid(s) and grown in YEP medium containing the indicated sugars. A shortened “pseudo-WT” (Ψ-WT) U6 allele is present in lanes 2–4 to provide U6 function in conditions where GAL-SNR6-II is repressed. Note that the level of U6-II is increased in the absence of Ψ-WT U6. U1 RNA serves as a normalization control.
