Herzog Veronika A, Reichholf Brian, Neumann Tobias, Rescheneder Philipp, Bhat Pooja, Burkard Thomas R, Wlotzka Wiebke, von Haeseler Arndt, Zuber Johannes, Ameres Stefan L
Institute of Molecular Biotechnology, Vienna Biocenter Campus, Vienna, Austria.
Research Institute of Molecular Pathology, Vienna Biocenter Campus, Vienna, Austria.
Nat Methods. 2017 Dec;14(12):1198-1204. doi: 10.1038/nmeth.4435. Epub 2017 Sep 25.
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (sU) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.
通过高通量测序进行的基因表达谱分析揭示了稳态下RNA种类的定性和定量变化，但掩盖了RNA转录、加工和衰变的细胞内动力学。我们开发了用于RNA代谢测序的硫醇（SH）连接烷基化方法（SLAM seq），这是一种基于正交化学的RNA测序技术，能够以单核苷酸分辨率检测RNA种类中4-硫尿苷（sU）的掺入情况。结合成熟的代谢RNA标记方案并与标准的低输入、高通量RNA测序方法相结合，SLAM seq能够在总RNA的背景下快速获取RNA聚合酶II依赖性基因表达动力学。我们在小鼠胚胎干细胞中验证了该方法，结果表明RNA聚合酶II依赖性转录输出与Oct4/Sox2/Nanog定义的增强子活性成比例，并且我们提供了由微小RNA和N-甲基腺苷启动的转录后基因调控途径介导的转录本特异性RNA周转的定量和机制证据。SLAM seq有助于以一种可及、经济高效且可扩展的方式剖析控制基因表达的基本机制。
