Grr1-mediated Ubp3 degradation is crucial for HAC1 mRNA translation and unfolded stress response in yeast [Riboseq]. Grr1-mediated Ubp3 degradation is crucial for HAC1 mRNA translation and unfolded stress response in yeast [Riboseq]

Ribosome ubiquitination induced by ribosome stalling is crucial for quality control pathways targeting mRNA, nascent polypeptides, and non-functional ribosomes. Besides quality control, Not4-mediated monoubiquitination of eS7A is crucial for the efficient translation of HAC1i mRNA in the unfolded protein response (UPR). In this study, we identified a novel E3 ligase, Grr1, an F-box protein component of the SCF ubiquitin ligase complex that is involved in Ubp3 degradation thereby HAC1i mRNA translation. Grr1 degrades Ubp3, a deubiquitinating enzyme of eS7A, under ER stress, thereby increasing eS7A ubiquitination, which facilitates HAC1i translation. Translation of HAC1i mRNA requires Grr1 and eS7 ubiquitination regardless of ER stress. ER stress-specific expression of Hac1 protein is ensured by the multi-step regulation of HAC1u mRNA, including localization on the ER membrane and stress-mediated splicing. Translation of HAC1i mRNA in UPR requires Grr1-mediated eS7 ubiquitination. More importantly, exon 1 of the HAC1i mRNA is crucial for translation activation by eS7 ubiquitination. Collectively, we propose that Grr1 upregulates eS7A monoubiquitination, thereby HAC1i translation and plays a crucial role in UPR. Overall design: To investigate how ER stress affects translation, we performed ribosome profiling (Ribo-seq) of tunicamycin-treated W303 strains. The dataset includes 4 types of Ribo-seq samples in biological replicates (indicated as rep1 and rep2; 8 samples in total). Samples differ by the genotype (wild-type or grr1 deficient), and tunicamycin treatment time (0, 4 hr). We then calculated Translation efficiency (TE) using the read counts from Ribo-seq data and the read counts from RNA-seq data shown in another file. Besides, we analyzed ribosome footprints throughout HAC1i mRNA. For each genotype (wild-type or grr1 deficient), 0 hr data was compared against data of 4 hr data.

核糖体停滞诱导的核糖体泛素化，对于靶向信使RNA（mRNA）、新生多肽链以及无功能核糖体的质量控制通路至关重要。除质量控制外，Not4介导的核糖体蛋白eS7A单泛素化，对于未折叠蛋白反应（unfolded protein response, UPR）中剪接型HAC1 mRNA（HAC1i mRNA）的高效翻译至关重要。本研究中，我们鉴定出一种新型E3泛素连接酶Grr1——它是SCF泛素连接酶复合物的F-box蛋白组分，参与Ubp3的降解进而调控HAC1i mRNA的翻译。在内质网应激条件下，Grr1降解eS7A的去泛素化酶Ubp3，从而提升eS7A的泛素化水平，促进HAC1i mRNA的翻译。无论是否存在内质网应激，HAC1i mRNA的翻译均依赖Grr1与eS7泛素化。Hac1蛋白的内质网应激特异性表达，通过对未剪接型HAC1 mRNA（HAC1u mRNA）的多步调控得以保障，这包括其在内质网膜上的定位以及应激介导的剪接过程。未折叠蛋白反应中HAC1i mRNA的翻译，依赖Grr1介导的eS7泛素化。更为关键的是，HAC1i mRNA的外显子1对于eS7泛素化介导的翻译激活至关重要。综上，我们提出：Grr1通过上调eS7A的单泛素化水平，进而促进HAC1i mRNA的翻译，在未折叠蛋白反应中发挥关键作用。实验设计概述：为探究内质网应激如何影响翻译过程，我们对衣霉素处理的W303菌株开展了核糖体足迹测序（ribosome profiling, Ribo-seq）。本数据集包含4种类型的核糖体足迹测序样本，均设置生物学重复（标记为rep1与rep2，总计8个样本）。样本的差异在于基因型（野生型或grr1缺陷型）以及衣霉素处理时长（0小时、4小时）。随后，我们利用核糖体足迹测序数据的读段计数，与另一文件中RNA测序（RNA-seq）数据的读段计数，计算得到翻译效率（translation efficiency, TE）。此外，我们还分析了HAC1i mRNA全长的核糖体足迹分布。针对每种基因型（野生型或grr1缺陷型），我们将0小时处理组的数据与4小时处理组的数据进行了比较。