Ribosome footprinting in the cytosol and endoplasmic reticulum. Homo sapiens

In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a role for the ER in global protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression. Overall design: HEK293 cells were fractionated between the cytosol and endoplasmic reticulum. Within each fraction, ribosome footprints were generated and sequenced. In parallel, total mRNA was sequenced.

真核细胞中，蛋白质表达的空间调控通常通过mRNA定位与翻译局部调控的协同耦合得以实现。mRNA定向定位至内质网（endoplasmic reticulum, ER）是这类调控的典型范例，其在将分泌蛋白与整合膜蛋白的合成定向分配至内质网的过程中发挥广泛作用。近年的基因组学与生物化学研究拓展了这一认知，表明内质网在全局蛋白质合成中亦具备功能性角色。本研究采用细胞分级分离技术与核糖体谱分析（ribosome profiling），对mRNA翻译的亚细胞组织模式开展了基因组范围的系统性调研，结果发现作为mRNA翻译替代指标的mRNA核糖体装载量，在内质网组分中呈现显著富集偏向性。值得注意的是，编码胞质蛋白与信号肽编码蛋白的内质网结合mRNA，其核糖体装载密度并无显著差异，这表明内质网结合核糖体在mRNA翻译过程中承担着全局性功能。本研究提出，mRNA及其翻译过程在胞质与内质网组分间的分区分配，或许代表了一种全新的基因表达转录后调控机制。实验整体设计：将HEK293细胞分级分离为胞质与内质网两个组分；对每个组分中的核糖体足迹（ribosome footprints）进行建库测序，同时并行开展总mRNA的建库测序。