RNA-蛋白质复合物规模化富集与鉴定新方法

A New Method for Large-scale Enrichment and Stepwise Identification of RNA-protein Complexes

RNA-蛋白质复合物调控着生物体内各种生理过程,在RNA从合成到降解的生理活动历程中发挥关键作用.为了更全面解析RNA-蛋白复合物(RNA-protein complexes,RPC)相互作用网络,近年来研究者除了开发RNA直接结合蛋白(RNA-binding proteins,RBP)规模化富集方法,也将目光转向了与RBP存在相互作用的RNA间接结合蛋白(RNA-associated proteins,RAP),二者与RNA共同组成了结构复杂、功能各异的RPC.但以往方法均将RPC中包含的所有蛋白视为整体,统一鉴定,缺乏能够规模化区分RBP和RAP的有效途径.基于此,本文联合使用254 nm紫外交联法和二硫双琥珀酰亚胺丙酸酯(DSP)蛋白交联剂,通过不同反应原理将RBP与RAP分别富集于链霉素磁珠,进而通过分步洗脱方法分别获取纯化的RBP和RAP,并结合质谱分析实现二者的各自独立鉴定.采用该方法在HeLa细胞中共鉴定到2007个高置信RBP,927个高置信RAP,其中有243个RAP为首次鉴定.此方法的建立有助于细化RPC中各组分间的相互作用网络,为后续的生物应用提供方法和数据基础.

RNA-protein complexes(RPC) regulate various physiological processes in organisms and play a crucial role in the synthesis and degradation of RNA.For deep RPC profiling in addition to develop RNA-binding proteins(RBP) enrichment methods,some researchers also turned their attention to RNA-associated proteins(RAP),which interact with RBP via protein-protein interaction.However,previous methods often enrich all proteins in the RPC as a whole and cannot differentiate RBP from RAP.Therefore,we reported a new method using combination of 254 nm UV crosslinking of RBP and RNA and DSP crosslinking of RBP and RAP.Next,RBP and RAP can be eluted in a stepwise way and separately identified by mass spectrometry.A total of 2007 high confidence RBP and 927 high confidence RAP were identified in HeLa cells,of which 243 RAP were identified for the first time.This method aims to refine the RNA-protein interaction network and provide a favorable foundation for subsequent biological investigations.