From the Department of Cardiology, Angiology, and Pneumology, Internal Medicine III, Heidelberg University Hospital (S.D., C.H., E.B., B.M., E.R., A.A.G., T.J., C.S., L.J., V.K., E.M., J. Burghaus, F.Y., H.A.K., C.D., M.V.).
DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany (S.D., C.H., E.B., B.M., E.R., A.A.G., T.J., C.S., L.J., V.K., E.M., J. Burghaus, F.Y., V.M., J. Backs, H.A.K., C.D., M.V.).
Circ Res. 2019 Aug 2;125(4):431-448. doi: 10.1161/CIRCRESAHA.119.314817. Epub 2019 Jul 9.
Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells.
This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload.
By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation.
This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.
基因表达谱主要通过分析转录物丰度来确定。然而，这些分析无法捕捉到翻译水平的转录后基因表达调控，因为转录物水平通常与蛋白质水平相关性较差，这证明了翻译是基因表达调控的关键步骤。此外，由于组织的裂解物总是代表多种细胞的混合物，因此对不同细胞类型进行全基因组转录谱分析具有挑战性。
本研究旨在开发一种新的实验方法来克服这两个限制，并应用该方法在压力超负荷时对翻译水平的基因表达进行全基因组分析。
通过将核糖体分析（Ribo-seq）与核糖体标记方法（Ribo-tag）相结合，我们能够从心脏中特定的细胞类型中确定翻译转录组。在压力超负荷后，我们通过从心脏裂解物中纯化标记的心肌细胞核糖体，并对核糖体保护的 mRNA 片段进行深度测序，监测心肌细胞的翻译组。我们鉴定出了在翻译水平受到调控的 mRNA 亚群，并发现翻译控制决定了心肌细胞对心脏应激的早期基因表达变化。翻译调控的转录本与与翻译、蛋白质质量控制和代谢相关的特定生物学过程相关联。从机制上讲，Ribo-seq 允许鉴定转录物中的上游开放阅读框，我们预测这些开放阅读框是翻译的重要调节剂。
该方法具有以下潜力：(1) 为在组织中研究细胞特异性翻译水平的基因表达提供新工具；(2) 揭示新的治疗靶点以预防细胞重塑；(3) 引发后续研究，既涉及心脏细胞中转录后基因表达调控的分子机制，也涉及细胞应激时表达的蛋白质的保护功能。
