Ribosome profiling upon glucose starvation in S. cerevisiae. Saccharomyces cerevisiae

A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes encoding proteins important for survival. Interestingly, under many of these conditions overall protein synthesis levels are reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in yeast, translation is rapidly and reversibly repressed, yet transcription of many stress- and glucose-repressed genes is increased. Using ribosome profiling and microscopy, we found that this transcriptionally upregulated gene set consists of two classes: (1) one producing mRNAs that are preferentially translated during glucose limitation and are diffusely localized in the cytoplasm – this class includes many heat shock protein mRNAs; and (2) another producing mRNAs that are poorly translated during glucose limitation, have high rates of translation initiation, and are concentrated in foci that co-localize with P bodies and stress granules – this class is enriched for glucose metabolism mRNAs. Remarkably, the information specifying differential localization and translation of these two classes of mRNAs is encoded in the promoter sequence – promoter responsiveness to heat shock factor (Hsf1) specifies diffuse cytoplasmic localization and preferential translation upon glucose starvation, whereas different promoter elements upstream of genes encoding poorly translated glucose metabolism mRNAs direct these mRNAs to RNA granules under glucose starvation. Thus, promoter sequences and transcription factor binding can influence not only mRNA levels, but also subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to environmental conditions. Overall design: Examination of mRNA translation in S. cerevisiae upon glucose starvation.

应激与营养限制应答的一个普遍特征是：对编码生存必需蛋白的基因进行转录上调。有趣的是，在多数此类条件下，整体蛋白质合成水平会降低，从而在蛋白质表达层面减弱应激应答。例如，在酿酒酵母（S. cerevisiae）葡萄糖饥饿过程中，翻译过程会被快速且可逆地抑制，但众多应激相关及葡萄糖抑制基因的转录水平却会升高。本研究通过核糖体谱分析（ribosome profiling）与显微镜成像技术，发现这类转录上调的基因集可分为两类：(1) 其转录出的mRNA在葡萄糖限制条件下优先被翻译，且在细胞质中呈弥散分布——该类别包含众多热休克蛋白mRNA；(2) 其转录出的mRNA在葡萄糖限制条件下翻译效率低下，但翻译起始速率较高，且会聚集在与处理小体（P bodies）及应激颗粒共定位的焦点中——该类别富含葡萄糖代谢相关mRNA。值得注意的是，决定这两类mRNA差异定位与翻译的信息编码于启动子序列中：对热休克因子（Hsf1）具有响应性的启动子，可指导mRNA在葡萄糖饥饿时呈弥散性胞质分布并获得优先翻译；而编码低翻译效率葡萄糖代谢基因的上游启动子元件，则会引导这些mRNA在葡萄糖饥饿时聚集至RNA颗粒中。由此可见，启动子序列与转录因子结合不仅可以调控mRNA的表达水平，还能影响mRNA的亚细胞定位及其翻译效率，使细胞能够根据环境条件调整蛋白质合成水平。实验设计：探究葡萄糖饥饿条件下酿酒酵母（S. cerevisiae）的mRNA翻译情况。