紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

Efficient Assay of The RNA-binding Activities in a Protein Complex by UV Cross-linking and Tandem Affinity Purification

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.

RNA-binding proteins （RBPs） function importantly in RNA synthesis and metabolism by interacting with specific RNAs. The identification of RNA-protein interactions is required for understanding the cellular function mechanisms. A new technique, PAR-CLIP （photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation）, has been used to determine the target transcripts of a few RBPs and map the RNA binding sites in a genome-wide fashion. In this study, a rapid and effective method was developed on the basis of PAR-CLIP to detect the RNA binding activity of putative RBPs and their complexes, termed UV cross-linking and tandem affinity purification （CLiTAP）. The improvements include： （1） Tandem affinity purification was performed to purify RNA-protein complexes efficiently; （2） Sypro Ruby staining and autoradiography were used sequentially to determine which protein（s） possessing RNA-binding activities. CLiTAP was used to analyze the RNA binding activity of three kinds of CCCH-type zinc finger proteins, TbZC3H7, TbZC3H34 and TbZC3H5, in Trypanosoma brucei. TbZC3H7, a core component of RNA cap-binding complex, showed a strong RNA-binding ability. TbZC3H34, a hypothetical protein, displayed a weak RNA-binding activity, while one of its interacting proteins showed a strong activity. In contrast, TbZC3H5 and all its interacting proteins did not show any RNA binding activity. These results indicated that CLiTAP is an efficient method and can be used to experimentally identify the RBPs in a protein complex, which provide a basis for further mapping the RNA binding sites and investigating the structure and mechanism of RBPs.