EMBL-Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia.
State Socio-Humanitarian University, Kolomna, Russia.
Nat Protoc. 2017 Apr;12(4):697-731. doi: 10.1038/nprot.2016.189. Epub 2017 Mar 2.
Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome-mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes ∼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.
信使 RNA（mRNA）翻译是一个受到严格调控的基因表达过程，涉及核糖体的小亚基和大亚基与 mRNA 之间复杂而动态的相互作用，在不同的起始、延伸和终止阶段，还需要多种辅助因子的参与。最近开发的核糖体图谱（ribosome profiling）方法可以对翻译及其调控进行全转录组范围的研究。核糖体图谱记录了完整核糖体在 mRNA 上的足迹，因此主要研究翻译的延伸阶段。重要的是，它不能监测涉及小核糖体亚基（SSU）和 mRNA 之间复合物的起始和终止的多个子步骤。在这里，我们描述了一种相关的方法，称为“翻译复合物图谱测序”（translation complex profile sequencing，TCP-seq），它独特地能够全转录组范围内记录任何类型核糖体-mRNA 复合物的位置。它使用活细胞中翻译复合物的快速共价固定，然后对翻译中间产物进行 RNase 足迹分析，并将其分离成涉及完整核糖体或 SSU 的复合物。然后分别对每种类型的复合物的足迹进行深度测序，生成在延伸以及起始和终止阶段翻译过程中的自然分布图谱。我们为酿酒酵母液体悬浮培养提供了完整的 TCP-seq 方案，包括数据分析流程。对于熟悉标准分子生物学技术、有超速离心和 RNA 测序（RNA-seq）文库制备经验的研究人员，完成该方案大约需要 3 周的时间。使用提供的生物信息学工具需要基本的 Bash 和 UNIX/Linux 命令技能。
