Nature Protocols ( IF 14.8 ) Pub Date : 2020-11-18 , DOI: 10.1038/s41596-020-00404-1 Joel I Perez-Perri 1 , Marko Noerenberg 2, 3 , Wael Kamel 2 , Caroline E Lenz 2 , Shabaz Mohammed 2, 4, 5 , Matthias W Hentze 1, 6 , Alfredo Castello 2, 3
Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues such as metabolic imbalance or virus infection. Enhanced RIC exploits the stronger binding of locked nucleic acid (LNA)-containing oligo(dT) probes to poly(A) tails to maximize RNA capture selectivity and efficiency, profoundly improving signal-to-noise ratios. The subsequent analytical use of SILAC and TMT proteomic approaches, together with high-sensitivity sample preparation and tailored statistical data analysis, substantially improves RIC’s quantitative accuracy and reproducibility. This optimized approach is an extension of the original RIC protocol. It takes 3 d plus 2 weeks for proteomics and data analysis and will enable the study of RBP dynamics under different physiological and pathological conditions.
中文翻译:
通过比较和增强的 RNA 相互作用组捕获对 RNA 结合蛋白动力学的全局分析
RNA 结合蛋白 (RBP) 和 RNA 之间的相互作用对细胞生物学至关重要。然而,缺乏全面和定量评估细胞内这些相互作用的方法。RNA 相互作用组捕获 (RIC) 使用体内 UV 交联、oligo(dT) 捕获和蛋白质组学来识别 RNA 结合蛋白质组。最近的进展使 RIC 能够量化 RBP 对代谢失衡或病毒感染等生物线索的反应。增强型 RIC 利用含锁定核酸 (LNA) 的寡 (dT) 探针与多聚 (A) 尾的更强结合来最大限度地提高 RNA 捕获选择性和效率,从而显着提高信噪比。随后对 SILAC 和 TMT 蛋白质组学方法的分析使用,以及高灵敏度样品制备和量身定制的统计数据分析,大大提高了 RIC 的定量准确性和重现性。这种优化的方法是原始 RIC 协议的扩展。蛋白质组学和数据分析需要 3 天加 2 周,这将使研究不同生理和病理条件下的 RBP 动力学成为可能。