相互作用体
核糖核酸
生物素化
计算生物学
蛋白质组
蛋白质组学
抄写(语言学)
生物
分子生物学
化学
RNA结合蛋白
生物化学
基因
语言学
哲学
作者
Xiangpeng Guo,Muqddas Tariq,Yiwei Lai,Shahzina Kanwal,Yuan Lv,Xiwei Wang,Na Li,Mengling Jiang,Jin Meng,Jieyi Hu,Jianwen Yuan,Zhiwei Luo,Carl Ward,Giacomo Volpe,Dongye Wang,Menghui Yin,Baoming Qin,Biliang Zhang,Xichen Bao,Miguel A. Esteban
出处
期刊:Nature Protocols
[Springer Nature]
日期:2021-10-25
卷期号:16 (11): 5193-5219
被引量:7
标识
DOI:10.1038/s41596-021-00609-y
摘要
Application of synthetic nucleoside analogues to capture newly transcribed RNAs has unveiled key features of RNA metabolism. Whether this approach could be adapted to isolate the RNA-bound proteome (RNA interactome) was, however, unexplored. We have developed a new method (capture of the newly transcribed RNA interactome using click chemistry, or RICK) for the systematic identification of RNA-binding proteins based on the incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV cross-linking and click chemistry-mediated biotinylation. The RNA-protein adducts are then isolated by affinity capture using streptavidin-coated beads. Through high-throughput RNA sequencing and mass spectrometry, the RNAs and proteins can be elucidated globally. A typical RICK experimental procedure takes only 1 d, excluding the steps of cell preparation, 5-ethynyluridine labeling, validation (silver staining, western blotting, quantitative reverse-transcription PCR (qRT-PCR) or RNA sequencing (RNA-seq)) and proteomics. Major advantages of RICK are the capture of RNA-binding proteins interacting with any type of RNA and, particularly, the ability to discern between newly transcribed and steady-state RNAs through controlled labeling. Thanks to its versatility, RICK will facilitate the characterization of the total and newly transcribed RNA interactome in different cell types and conditions.
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