构建MS2-RIP方法用于鉴定lncRNA DANCR在肿瘤细胞中的相互作用分子

MS2-RIP Assay for Identifying the Interaction Partners of lncRNA DANCR in Tumor Cells

【目的】为了探讨lncRNA DANCR在肿瘤细胞中的作用机制,本研究拟构建一种新的MS2-RIP方法用于鉴定DANCR的相互作用分子。【方法】利用MS2噬菌体衣壳蛋白可与噬菌体复制酶编码基因5’端一段由19个碱基组成的RNA茎环结构序列,即MS2结合位点(MS2 Binding Site,MS2 BS)高效结合的特点,构建串联表达MS2 BS和DANCR的过量表达质粒,通过MS2蛋白与MS2 BS的亲和作用,使DANCR得到富集,并且同步捕获与DANCR相互作用的分子,用于进一步分析鉴定。【结果】成功构建串联表达MS2 BS和DANCR的过量表达质粒用于MS2-RIP实验,该实验可显著富集DANCR以及作为阳性对照的DANCR结合蛋白EZH2。【结论】基于MS2蛋白与MS2结合位点的高效结合特性,我们针对lncRNA DANCR建立了MS2-RIP方法。该方法可有效富集DANCR以及与DANCR结合的生物活性分子,为研究DANCR的作用机制提供一种新的技术手段。

【Objective】To explore the functional mechanism of lncRNA DANCR in tumor cells,a MS2-RIP method was designed and conducted to identify the molecules that interact with DANCR.【Methods】The specific binding of MS2 bacteriophage capsid protein and MS2 binding site(MS2 BS),a 19-base RNA stem-loop structure located at the 5'terminus of the MS2 bacteriophage replicase gene,was applied to construct a plasmid for tandemly overexpressing DANCR and MS2 BS.DANCR was enriched and its associated molecules were further identified and analyzed.【Results】The plasmid for tandemly overexpressing DANCR and MS2 BS was successfully constructed for the MS2-RIP experiment,which could significantly enrich DANCR and DANCR-binding protein EZH2 as a positive control.【Conclusion】Based on the high affinity binding between MS2 protein and MS2 BS,the MS2-RIP method was established for lncRNA DANCR,which could effectively capture DANCR as well as its associated molecules,providing a new technology for studying the functional mechanism of DANCR.