Fig. 7: Grr1 is involved in efficient HAC1i translation via eS7A ubiquitination.
From: Crucial roles of Grr1 in splicing and translation of HAC1 mRNA upon unfolded stress response
A 3xFLAG-Hac1p/CBB and 3xFLAG-GFP/CBB levels shown in Supplementary Fig. 9A were quantified and normalized protein levels relative to that in WT. B 3xFLAG-Hac1p expressions are decreased in grr1∆eS7A(K83 only), eS7A(K83R) and grr1∆eS7A(K83R). Yeast cells harboring plasmids encoding 3xFLAG-HCt under control of TEF1 promoters were grown at 30 °C and harvested at log phase. 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%. C 3xFLAG-Hac1p/CBB levels shown in (B) and Supplementary Fig. 9B were quantified and normalized protein levels relative to that in eS7A(K83 only). Data represent n = 3 biologically independent experiments (mean ± SE). D 3xFLAG-Hac1p expressions are decreased in grr1∆, but not in grr1∆ubp3∆. Yeast cells harboring plasmids encoding 3xFLAG-HCt under control of TEF1 promoters were grown at 30°C and harvested at log phase. The samples were analyzed as in (B). E 3xFLAG- Hac1p/CBB levels shown in (D) and Supplementary Fig. 9C were quantified and normalized protein levels relative to that in WT. Data represent n = 4 (WT, grr1∆) or 8 (ubp3∆, grr1∆ubp3∆) biologically independent experiments (mean ± SE). F Schematic drawings of T7 promoter−2xPA-(HAC1i or GFP)-polyA70 mRNAs. G 2xPA-Hac1 translation is decreased in grr1∆ski2∆ lysate. In vitro translation was performed using 2xPA-HAC1i-polyA70 and 2xPA-GFP-polyA70 mRNAs at 17 °C for 30 min. The samples were analyzed using western blotting with anti-PA antibody. Dilutions are 100%, 75%, 50%, 25%, 12.5%, 6.25%. H 2xPA-Hac1/2xPA-GFP levels shown in (G) were quantified and normalized protein levels relative to that in ski2∆ lysate. I Model for the crucial role of Grr1-mediated degradation of Ubp3 in the translation of HAC1i mRNA. Not4-mediated monoubiquitination of eS7A is required for efficient translation of HAC1i mRNA. The SCFGrr1 complex is responsible for the downregulation of Ubp3, which facilitates eS7A ubiquitination and HAC1i translation. All experiments were repeated at least twice with biologically independent samples and showed similar results. Data in (A, H) represent n = 4 biologically independent experiments (mean ± SE). P values were calculated by Two-sided Welch’s t-test. Source data are provided as a Source Data file.
