Cargando…

Acrylonitrile‐Mediated Nascent RNA Sequencing for Transcriptome‐Wide Profiling of Cellular RNA Dynamics

RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, p...

Descripción completa

Detalles Bibliográficos
Autores principales: Chen, Yuqi, Wu, Fan, Chen, Zonggui, He, Zhiyong, Wei, Qi, Zeng, Weiwu, Chen, Kun, Xiao, Feng, Yuan, Yushu, Weng, Xiaocheng, Zhou, Yu, Zhou, Xiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175251/
https://www.ncbi.nlm.nih.gov/pubmed/32328408
http://dx.doi.org/10.1002/advs.201900997
Descripción
Sumario:RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile‐mediated uridine‐to‐cytidine (U‐to‐C) conversion (AMUC‐seq) via 4‐thiouridine (s(4)U) cyanoethylation is presented. This method converts s(4)U base‐pairing with guanine through the nucleophilic addition of s(4)U to acrylonitrile. The high reaction efficiency permits AMUC‐seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC‐seq is validated by being used to detect mRNA half‐lives and investigating the direct gene targets of a G‐quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC‐seq also confirms G‐quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC‐seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription‐modulating drugs.