Monitoring Cell-Type-Specific Gene Expression Using Ribosome Profiling In Vivo During Cardiac Hemodynamic Stress
Monitoring Cell-Type-Specific Gene Expression Using Ribosome Profiling In Vivo During Cardiac Hemodynamic Stress
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DOI：
10.1161/circresaha.119.314817
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发表时间：
2019-08-02
影响因子：
20.1
通讯作者：
Voelkers, Mirko
中科院分区：
文献类型：
--
作者：
Doroudgar, Shirin;Hofmann, Christoph;Voelkers, Mirko
基本原理：基因表达谱主要通过分析转录本丰度来确定。然而，这些分析不能捕获在翻译水平上的转录后基因表达控制，这是基因表达调控中的关键步骤，如转录水平通常与蛋白质水平相关性差的事实所证明的。此外，由于来自组织的裂解物总是代表细胞的混合物，不同细胞类型的全基因组转录物谱分析是具有挑战性的。目的：本研究的目的是开发一种新的实验方法，克服这两个限制，并应用这种方法来进行全基因组分析的基因表达的翻译水平上的压力过载。方法和结果：通过将核糖体分析（Ribo-seq）与核糖体标记方法（Ribo-tag）相结合，可以确定来自心脏的特定细胞类型中的翻译转录组。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片段进行深度测序来监测心肌细胞翻译组。我们确定了在翻译水平上受调控的mRNA亚群，发现翻译调控决定了心肌细胞对心脏应激反应中基因表达的早期变化。翻译控制转录物与翻译、蛋白质质量控制和代谢相关的特定生物过程相关。从机制上讲，Ribo-seq允许识别转录本中的上游开放阅读框架，我们预测这是翻译的重要调节因子。结论：该方法有可能（1）提供用于在组织中翻译水平上研究细胞特异性基因表达的新工具，（2）揭示新的治疗靶点以防止细胞重塑，以及（3）引发针对以下两个方面的后续研究：参与心脏细胞中基因表达的转录后控制的分子机制，以及响应细胞应激而表达的蛋白质的保护功能。
Rationale: 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. Objectives: 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. Methods and Results: 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. Conclusions: 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.