Acrylonitrile-mediated nascent RNA sequencing for transcriptome-wide profiling of RNA dynamics

RNA sequencing has greatly facilitated gene expression studies but are weak in studying temporal RNA dynamics. This issue can be addressed by profiling nascent RNAs. Here, we presented a NGS-compatible method for nascent RNA analysis in single-base resolution based on s4U cyanoethylation reaction. Our strategy can high-efficiently recover nascent RNA information from total RNAs. We validated our method by measuring RNA stabilities transcriptome wide in human cells. By capturing immediate gene expression changes, we successfully determined the direct gene targets of a G-quadruplex stabilizer, which has been regarded as potential antitumor drug. We also identified the influence of G-quadruplex stabilization to mRNA maturation. Furthermore, we found G-quadruplex stabilizer positively regulated the transcriptional activity of upstream antisense RNAs, uncovering its novel potential gene regulation pathway in vivo. Overall design: total mRNA profiles of 4sU-labeled HEK293T cells (PDP-treated or PDP-untreated) were generated by deep sequencing, in duplicate, using HiSeq X Ten.RNA测序 (RNA sequencing) 极大地促进了基因表达研究，但在时序性RNA动态变化的分析中存在明显局限。针对这一问题，可通过新生RNA (nascent RNA) 谱分析予以解决。本研究开发了一种基于s4U氰乙基化反应、兼容下一代测序 (Next-Generation Sequencing, NGS) 的单碱基分辨率新生RNA分析方法，可从总RNA中高效回收新生RNA的相关信息。我们通过在人类细胞中开展全转录组水平的RNA稳定性检测，验证了该方法的有效性。通过捕获即时发生的基因表达变化，我们成功鉴定出一种被视为潜在抗肿瘤药物的G-四链体 (G-quadruplex) 稳定剂的直接基因靶标。此外，我们还揭示了G-四链体稳定化对信使RNA (messenger RNA, mRNA) 成熟过程的调控影响。进一步研究发现，G-四链体稳定剂可正向调控上游反义RNA的转录活性，由此揭示了其在体内发挥作用的新型潜在基因调控通路。 总实验设计：采用HiSeq X Ten测序平台，以双生物学重复的方式对经4sU标记的HEK293T细胞（经PDP处理组与未处理组）的总mRNA进行深度测序，获取其转录组表达谱。