Fig. 6: UTRs of HAC1 are not the target for eS7A ubiquitination-mediated translational regulation.

A Schematic drawing of 3xFLAG-HAC1ⁱ and 3xFLAG-GFP mRNAs. B Tunicamycin sensitivity of hac1∆ cells expressing 3xFLAG-HAC1ⁱ reporters. 10-fold serial dilutions of the indicated cells were spotted onto the plates. Plates were incubated at 30 °C for 2–3 days. C 3xFLAG-Hac1p expression is increased by eS7A ubiquitination independent of UTRs of HAC1. Yeast cells harboring plasmids encoding 3xFLAG-HAC1ⁱ shown in (A) under the control of TEF1 promoters were harvested as in Fig. 1B. The samples were analyzed using western blotting with anti-FLAG antibody. CBB staining was used as a loading control. Dilutions are 100%, 75%, 50%, 25%, 12.5%, 6.25%. D The 3xFLAG-Hac1p/CBB levels shown in (C) and Supplementary Fig. 8D were quantified and normalized relative to that from eS7A(WT) expressing 3xFLAG-5H3. The percentages indicate the protein levels ratio in eS7A(4KR) to that in eS7A(WT). E 3xFLAG-HAC1ⁱ mRNA levels in eS7A(WT) and eS7A(4KR). Yeast cells were harvested, and the samples were analyzed as in Fig. 5G, using DIG-labeled 3xFLAG probe. F 3xFLAG-HAC1ⁱ mRNA/25S rRNA levels shown in (E) and Supplementary Fig. 8E were quantified and normalized as in (D). G 3xFLAG-GFP expressions are almost the same between eS7A(WT) and eS7A(4KR). Yeast cells expressing 3xFLAG-GFPCt by TEF1 promoters were harvested, and the samples were analyzed as in (C). H The 3xFLAG-GFP/CBB levels shown in (G) were quantified and normalized relative to that from eS7A(WT). I Proteasome-dependent degradation of 3xFLAG-Hac1p in eS7A(WT) and eS7A(4KR). Yeast cells harboring plasmids encoding 3xFLAG-HCt under control of TEF1 promoter were grown at 30 °C until OD₆₀₀ = 0.2, then treated with 1 µg/mL of Tm and 50 µM of MG132 for 2 h and harvested. The samples were analyzed as in (C). J The 3xFLAG-Hac1p/CBB levels shown in (I) were quantified and normalized relative to that in eS7A(WT) with 50 µM MG132 treatment. All experiments were repeated at least twice with biologically independent samples and showed similar results. Data represent n = 3 biologically independent experiments (mean ± SE), and P-values were calculated by Two-sided Welch’s t-test. Source data are provided as a Source Data file.