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.