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Abstract
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。
Highlights
Generally, the enrichment strategies can be classified into two major categories: in vitro and in vivo. It has been successfully applied in many studies, the in vitro transcribed bait RNA lacks modifications or structural similarity compared with its natural counterpart
We summarize recent in vivo methodological advances in the large⁃ scale enrichment and identification of RP⁃complexes, including cross⁃linking and immunopre⁃ cipitation ( CLIP) and related methods, click chemistry⁃assisted methods, and organic phase separations
CLIP involves irradiating living cells with 254⁃nm ultraviolet ( UV) light to establish covalent bonds between RNA and proteins. This enables CLIP to purify RNAs bound to a specif⁃ ic RBP under conditions that are stringent enough to prevent co⁃purification of nonspecifically bound proteins or free RNAs
Summary
Weak interactions between the non⁃covalently bound proteins and RNA require mild washing to remove non⁃specific binding, which needs careful optimization. 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.
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