Fig. 5: eS7A ubiquitination is required for efficient Hac1p expression from HAC1ⁱ.

A Schematic drawing of HAC1^u mRNA and 5H3(HAC1-intronless) mRNA. B Hac1p expression from 5H3 reporters with different promoters. Cells were grown and harvested and the samples were analyzed as in Fig. 1B. C Induction of 5H3 from various promoters by Tm treatment. Yeast cells were grown at 30 °C until OD₆₀₀ = 0.2, then treated with 1 µg/mL of Tm for ~ 2 h and harvested. The samples were analyzed by northern blotting with DIG-labeled HAC1 probe. Methylene Blue staining was used as a loading control. Dilutions are 100%, 75%, 50%, 25%. D Fold change of 5H3 mRNA/25S rRNA levels shown in Fig. 5C and Supplementary Fig. 7A, B were quantified and normalized relative to that at 0 min samples. E The correlation between Hac1p levels and tunicamycin resistance. 10-fold serial dilutions of OD₆₀₀ = 0.3 cells grown in SDC-Ura medium were spotted onto SDC-Ura plates. Plates were incubated at 30 °C for 2–3 days. F eS7A ubiquitination is required for efficient Hac1p expression from 5H3 clones under control of HAC1 or TEF1 promoters. The indicated cells were harvested as in (C). The samples were analyzed as in Fig. 1B. G HAC1ⁱ and 5H3 mRNA levels in eS7A(WT) and eS7A(4KR). Yeast cells were harvested, and the total RNA samples were analyzed using northern blotting as in (C). H 5H3 mRNA/25S rRNA levels shown in (G) and Supplementary Fig. 7C were quantified and normalized relative to 2 h in hac1∆ encoding HAC1p-5H3. I Grr1 is required for efficient Hac1p expression from 5H3 under control of HAC1 or TEF1 promoters. Cells were harvested as in (C), and samples were analyzed as in (F). J HAC1ⁱ and 5H3 mRNA levels in WT and grr1∆. Yeast cells were harvested, and the samples were analyzed as in (C). K 5H3 mRNA/25S rRNA levels shown in (J) and Supplementary Fig. 7D were quantified and normalized relative to 2 h in WT. All experiments were repeated at least twice with biologically independent samples and showed similar results. Data represent n = 3 biologically independent experiments (mean ± SE). Source data are provided as a Source Data file.