eIF3g binding to GUCG boxes spread in mRNA coding regions mediates translational heat shock response

Heat shock response (HSR) is crucial for life's adaptation to heat. Here, we discovered a novel mechanism of HSR controlled at the translational level, which depends on the multi-subunit translation initiation factor, eIF3. The cis-element for this regulation is called GUCG box and occurs degenerately within protein-coding regions of regulated mRNAs. Being positioned at ribosome leading-edge through ?-propeller subunit eIF3i, the RNA-binding subunit eIF3g binds the motif and stabilizes mRNA anchoring during translation under mild heat. The eIF3g/i-regulated HSR consists of 64 genes including heat shock protein genes, engaging a quarter of the cellular ribosomes. The 5'-terminal coding regions of the HSR mRNAs are enriched with more GUCG boxes, some of which are masked by weak secondary structures. This arrangement enables eIF3g/i-dependent enhancement in part through melting inhibitory secondary structures. GUCG boxes spread evenly across the entire protein-coding regions of strongly translated mRNAs ~20 nt. apart, as if to prevent ribosome collision. Thus, eIF3 not only stabilizes initiating ribosomes at its leading-edge but may contribute to anchoring elongating ribosomes throughout coding regions under heat insult. Overall design: SELEX was performed for 12 rounds in each experiment, as described in 5, with 18 base-long randomized ribonucleotide sequence flanked by primer binding sites designed for cDNA synthesis, PCR and RNA synthesis by T7 RNA polymerase. For these experiments we used His6-tagged eIF3g/Tif35p or its complex with (untagged) eIF3i/Tif34p. The DNA fragments encoding them were amplified by PCR using budding yeast Saccharomyces cerevisiae genomic DNA and then cloned into pET28b (with hexa-histidine tag fused to its N-terminus, in the case of eIF3g) and were co-expressed in E. coli strain BL21-CodonPlus (DE3)-RILP (Stratagene). The expressed protein(s) was purified by Ni-NTA (Qiagen) and Q-sepharose (Cytiva).

热休克反应（Heat Shock Response, HSR）是生命适应高温环境的核心调控机制。本研究发现了一种全新的、受翻译水平调控的热休克反应机制，该机制依赖于多亚基翻译起始因子eIF3（eukaryotic translation initiation factor 3, eIF3）。该调控的顺式作用元件被命名为GUCG盒（GUCG box），其在受调控mRNA的蛋白质编码区中以简并形式存在。RNA结合亚基eIF3g通过eIF3i的β螺旋桨（β-propeller）结构域定位于核糖体前沿，结合该基序并在温和热应激条件下维持翻译过程中mRNA的锚定稳定性。受eIF3g/i调控的热休克反应涉及64个基因（包括热休克蛋白基因），其占用了细胞内四分之一的核糖体。热休克反应相关mRNA的5'端编码区富集了更多的GUCG盒，其中部分元件可被较弱的二级结构所遮蔽。这种排布方式使得eIF3g/i介导的增强效应可部分通过解聚抑制性二级结构来实现。GUCG盒在翻译活性较强的mRNA的整个蛋白质编码区中均匀分布，间距约为20 nt，仿佛可防止核糖体碰撞。因此，eIF3不仅可稳定前沿处的起始核糖体，还可能在热应激条件下协助锚定编码区全程中的延伸核糖体。整体实验设计：每轮实验均进行12轮指数富集的配体系统进化技术（Systematic Evolution of Ligands by Exponential Enrichment, SELEX），具体方法参照文献[5]；实验采用侧翼带有引物结合位点的18碱基长度的随机核糖核苷酸序列，该位点可用于cDNA合成、聚合酶链式反应（Polymerase Chain Reaction, PCR）以及T7 RNA聚合酶介导的RNA合成。本实验使用了带有His6标签的eIF3g/Tif35p，或其与（无标签）eIF3i/Tif34p形成的复合物。以酿酒酵母（Saccharomyces cerevisiae）基因组DNA为模板，通过PCR扩增编码上述蛋白的DNA片段，随后将其克隆至pET28b载体（针对eIF3g，其N端融合六组氨酸标签），并在大肠杆菌BL21-CodonPlus (DE3)-RILP菌株（Stratagene公司）中进行共表达。表达的蛋白通过Ni-NTA亲和层析柱（Qiagen公司）与Q-sepharose阴离子交换层析柱（Cytiva公司）进行纯化。