(a) Northern blot analysis of total RNA extracted from wild type and Ire1Δ cells untreated or treated with tunicamycin (1 µg/ml), and hybridized with a probe complementary to the ORF of Bip1 mRNA. Right panel: quantitation normalized to Pgk1 mRNA. (b) The abundance of a GFP mRNA driven by the Bip1 promoter (black) compared to endogenous Bip1 mRNA was determined as a time course after DTT (2 mM) addition by quantitative Northern blotting. (c) Wild type cells bearing a construct encoding the Nmt1 5′ UTR, Bip1 ORF and Bip1 3′ UTR driven by the Nmt1 promoter were pre-treated with tunicamycin (0.25 µg/ml, 1 hr). At different time points after thiamine (15 µM) addition (to effect transcriptional shut-off of the Nmt1 promoter), RNA was extracted and analyzed by Northern hybridization. Blots were probed for the Nmt1 5′ UTR. Nmt1-Bip1 mRNA and Nmt-tBip1 mRNA were quantitated and normalized to the unspecific band (asterisk). (d) RNA-sequence read density map of the Bip1 locus derived from mRNA-enriched (ribosome depleted) RNA in wild type cells untreated or treated with DTT (2 mM DTT, 1 hr; left panels). Data are representative of one of two biological replicates. Single nucleotide resolution of the 3′ terminus of Bip1 mRNA determined by 3′ RACE (right panels). (e) Mutational analysis of the Bip1 mRNA cleavage site by Northern blotting. Total RNA was extracted from wild type, Ire1Δ or cells carrying mutations in the Bip1 3′ UTR mRNA. Cells were treated with 2 mM DTT, 1 hr or left untreated as indicated. The fold-changes indicate Bip1 mRNA abundance relative to that of Pgk1 mRNA. (f) Strand-specific, mRNA enriched (after removal of ribosomal RNA) deep-sequence analysis of annotated ORFs (y-axis) compared to strand-specific polyA+ enriched mRNA deep-sequence analysis of annotated ORFs (x-axis) (see Figure 1—source data). The plot indicates the ratio of transcript abundance in unstressed vs DTT-stressed (2 mM DTT, 1 hr) wild type cells. Symbol sizes and colors are as described in Figure 1c.