Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.
Department of Bioengineering, University of California San Diego, La Jolla, CA, 92093, USA.
J Microbiol. 2020 Mar;58(3):217-226. doi: 10.1007/s12275-020-9536-2. Epub 2020 Jan 28.
Gene expression changes in response to diverse environmental stimuli to regulate numerous cellular functions. Genes are expressed into their functional products with the help of messenger RNA (mRNA). Thus, measuring levels of mRNA in cells is important to understand cellular functions. With advances in next-generation sequencing (NGS), the abundance of cellular mRNA has been elucidated via transcriptome sequencing. However, several studies have found a discrepancy between mRNA abundance and protein levels induced by translational regulation, including different rates of ribosome entry and translational pausing. As such, the levels of mRNA are not necessarily a direct representation of the protein levels found in a cell. To determine a more precise way to measure protein expression in cells, the analysis of the levels of mRNA associated with ribosomes is being adopted. With an aid of NGS techniques, a single nucleotide resolution footprint of the ribosome was determined using a method known as Ribo-Seq or ribosome profiling. This method allows for the high-throughput measurement of translation in vivo, which was further analyzed to determine the protein synthesis rate, translational pausing, and cellular responses toward a variety of environmental changes. Here, we describe a simple analysis pipeline for Ribo-Seq in bacteria, so-called simple translatome analysis tool for Ribo-Seq (STATR). STATR can be used to carry out the primary processing of Ribo-Seq data, subsequently allowing for multiple levels of translatome study, from experimental validation to in-depth analyses. A command-by-command explanation is provided here to allow a broad spectrum of biologists to easily reproduce the analysis.
基因表达的变化可以响应各种环境刺激，从而调节许多细胞功能。基因在信使 RNA（mRNA）的帮助下表达为其功能产物。因此，测量细胞中 mRNA 的水平对于了解细胞功能非常重要。随着下一代测序（NGS）的发展，通过转录组测序已经阐明了细胞中 mRNA 的丰度。然而，一些研究发现，翻译调控会导致 mRNA 丰度和蛋白质水平之间存在差异，包括核糖体进入和翻译暂停的速度不同。因此，mRNA 的水平不一定能直接反映细胞中存在的蛋白质水平。为了更精确地测量细胞中的蛋白质表达水平，人们正在采用分析与核糖体相关的 mRNA 水平的方法。借助 NGS 技术，使用称为 Ribo-Seq 或核糖体分析的方法，确定了核糖体的单核苷酸分辨率足迹。该方法允许高通量测量体内翻译，随后对其进行分析，以确定蛋白质合成率、翻译暂停以及细胞对各种环境变化的反应。在这里，我们描述了一种用于细菌的 Ribo-Seq 的简单分析流程，即所谓的用于 Ribo-Seq 的简单翻译组分析工具（STATR）。STATR 可用于对 Ribo-Seq 数据进行初步处理，随后允许进行多个翻译组研究层次，从实验验证到深入分析。这里提供了一条命令一条命令的解释，以便广泛的生物学家能够轻松重现分析。
