Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Institute for Genetics, University of Cologne, Cologne, Germany.
Nat Protoc. 2019 May;14(5):1603-1633. doi: 10.1038/s41596-019-0152-8. Epub 2019 Apr 24.
RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression.
RNA 降解确保了细胞内 mRNA 转录本的适当水平，并消除了异常的 RNA。由于涉及的酶具有高度的连续性，降解中间产物由于固有不稳定性，因此对 RNA 降解动力学的详细研究迄今为止是不可行的。为了可视化衰减中间体并表征 mRNA 衰减的时空动力学，我们开发了一套应用 XRN1 抗性 RNA 序列（xrRNA）的方法，以保护 mRNA 转录本免受 5'-3'外切核酸酶消化。据我们所知，这种方法是唯一可以检测 mRNA 降解方向性并允许在未受干扰的细胞中跟踪降解产物的方法。在这里，我们提供了 xrRNA 报告基因设计、转染和细胞系生成的详细步骤。我们解释了如何从哺乳动物细胞中提取 xrRNA 报告基因 mRNA，以及如何使用 northern blot 和定量 PCR 检测和定量。该过程进一步侧重于如何使用单分子荧光显微镜检测和定量完整的报告基因 mRNA 和 XRN1 抗性降解中间产物。它提供了使用固定细胞和活细胞进行样品制备和图像采集的详细说明。该过程特别强调了详细描述高通量图像分析管道，该管道与文章一起提供，并旨在执行斑点共定位、检测效率归一化以及必要的结果解释质量控制步骤。这里发布的分析软件的目的是使非专业读者能够在报告基因 mRNA 表达后 4-6 天内检测和定量 RNA 衰减中间产物。
