Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada.
Methods. 2018 Mar 15;137:90-98. doi: 10.1016/j.ymeth.2017.12.006. Epub 2017 Dec 13.
Every step in the life cycle of an RNA transcript provides opportunity for regulation. One important aspect of post-transcriptional control is the regulation of RNA stability. Of the many strategies for determining mRNA stability, transcription inhibition and metabolic labeling have proved the most amenable to high-throughput analysis and have opened the door to dissecting mRNA decay transcriptome-wide. Here, we describe experimental and computational methods to determine transcriptome-wide RNA stabilities using both pharmacological inhibition of transcription and metabolic labeling. To aid in the analysis of these experiments, we discuss key characteristics of high-quality experiments and address other experimental and computational considerations for the analysis of mRNA stability. Broader application of these approaches will further our understanding of mRNA decay and illuminate its contribution to different biological processes.
RNA 转录本生命周期的每一步都提供了调控的机会。转录后调控的一个重要方面是 RNA 稳定性的调控。在确定 mRNA 稳定性的许多策略中，转录抑制和代谢标记已被证明最适合高通量分析，并为全面剖析 mRNA 衰变打开了大门。在这里，我们描述了使用转录抑制和代谢标记来确定全转录组 RNA 稳定性的实验和计算方法。为了帮助分析这些实验，我们讨论了高质量实验的关键特征，并解决了用于分析 mRNA 稳定性的其他实验和计算考虑因素。更广泛地应用这些方法将增进我们对 mRNA 衰变的理解，并阐明其对不同生物学过程的贡献。
