核糖核酸-蛋白质复合物规模化富集与鉴定技术的研究进展  

Advances in technologies for large-scale enrichment and identification of ribonucleic acid-protein complexes

作　　者：樊智雅 秦伟捷 FAN Zhiya;QIN Weijie(Beijing Institute of Lifeomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), State Key Laboratory of Proteomics, Beijing 102206, China)

机构地区：[1]军事科学院军事医学研究院生命组学研究所,北京蛋白质组研究中心,国家蛋白质科学中心(北京),蛋白质组学国家重点实验室,北京102206

出　　处：《色谱》2021年第2期105-111,共7页Chinese Journal of Chromatography

基　　金：国家重点研发计划(2016YFA0501403);国家自然科学基金(21675172).

摘　　要：核糖核酸(RNA)在细胞中并非单独存在,从它们产生到被降解的过程中与大量蛋白质发生相互作用,RNA结合蛋白(RNA-binding proteins,RBPs)能与RNA结合形成RNA-蛋白质复合物(RP复合物),并以这种复合物的形式发挥生理功能。RNAs或RBPs任一组分的异常与缺失都会影响RP复合物的正常生理功能,从而导致疾病的发生,如代谢异常、肌肉萎缩症、自身免疫性疾病和癌症。因此,定性定量分析RBPs及其在正常细胞和肿瘤细胞中与RNAs靶标之间的复杂相互作用网络有助于挖掘RP复合物在肿瘤发生发展中的作用,开发肿瘤生物标志物和新的治疗方式。要深入研究和理解RNAs与RBPs的相互作用网络,须依赖组学技术对RP复合物进行大规模鉴定。而作为在组学层面系统性解析RP复合物组成、含量和功能的第一步,大规模富集RP复合物极具挑战性。为了解决这一难题,研究者们发展了各种富集鉴定策略。该文针对RP复合物富集策略的最新进展进行了综述,包括紫外光交联和免疫沉淀(crosslinking and immunoprecipitation,CLIP)及其衍生技术、基于“点击化学”的富集策略和基于相分离的富集策略,比较分析了它们的技术原理、优缺点,以方便研究者们选择合适的策略来解决感兴趣的生物学问题。该文最后总结了当前的RP复合物富集方法仍然存在富集效率低和操作繁琐等亟需解决的技术挑战,为富集策略的发展提供了研究方向。Ribonucleic acid(RNA)rarely exists alone in the cell.RNAs interact with a variety of proteins and form RNA-protein complexes(RP-complexes)in every step of their life cycle,from transcription to degradation.These RP-complexes play key roles in regulating a variety of physiological processes.Defects in the composition and function of RP-complexes have been associated with many diseases,including metabolic disorders,muscular atrophy,autoimmune diseases,and cancer.It is hence evident that deciphering the highly complex interaction network of RNA-binding proteins(RBPs)and their RNA targets will provide a better understanding of disease development and lead to the discovery of new targets for cancer therapy.Large-scale identification of RP-complexes at the omics level is a prerequisite for obtaining insights into the complex RNA-protein interaction network.As the first step in omics-wide decoding of RP-complexes,enrichment and purification of RP-complexes is a highly challenging task.Recently,intensive efforts have been undertaken to better enrich and identify RP-complexes.Generally,the enrichment strategies can be classified into two major categories:in vitro and in vivo.Although it has been successfully applied in many studies,the in vitro transcribed bait RNA lacks modifications or structural similarity compared with its natural counterpart.Further,since the proteins relocate and remodel after cell lysis,the use of cell lysates as a protein source may result in capturing false interacting proteins that bind non-physiologically with the bait RNA.Finally,weak interactions between the non-covalently bound proteins and RNA require mild washing to remove non-specific binding,which needs careful optimization.However,substantial sample loss is inevitable.To overcome the disadvantages of in vitro approaches,in vivo cross-linking strategies that“freeze”natural RNA-protein complexes in intact cells via covalent cross-linking have become increasingly popular.The in vivo methods allow RNA to interact with proteins in the in

关 键 词：核糖核酸结合蛋白 紫外光交联和免疫沉淀 规模化富集 生物正交反应 相分离 

分 类 号：O658[理学—分析化学]