转录中介体与非编码RNA相互作用的功能与机理研究

It was recently suggested that non-coding RNAs (ncRNAs) may participate gene regulation through the Mediator complex. However, the functional and mechanistic relationship between Mediator and ncRNAs are not well understood. Here we plan to screen and identify the ncRNAs that specifically bind to the Mediator. We have previously showed that Mediator MED23 participate the transcription elongation and mRNA processing. We recently found that MED23 protein coelutes with RNAs, and bioinformatic analysis revealed a NusB-like RNA-binding domain within the MED23. RNA-immunoprecipitation (RIP) experiment was performed, identifying Mediator –associated ncRNAs in a MED23-dependent manner. We plan to use CLIP-seq to verify the interaction between MED23 and ncRNAs; and to dissect the RNA-binding motif within the MED23 using gene editing method. These studies will reveal the novel regulatory roles of both Mediator and ncRNAs.

有证据表明非编码RNA(ncRNA)可能通过转录中介体（Mediator）参与基因的表达调控，但其分子机理还有待于深入研究。我们的研究发现，中介体MED23亚基参与基因转录的起始、延伸和mRNA加工；最近我们在纯化中介体过程中又发现MED23蛋白与核酸在分子筛中共同洗脱；通过生物信息学分析发现MED23亚基的C端存在一个与细菌NusB蛋白同源的RNA结合结构域；RNA-seq结果表明MED23亚基可能结合ncRNA。我们拟运用MED23敲除的细胞系，结合CLIP-seq技术，筛选和鉴定可能与MED23特异结合的ncRNA，验证其特异的相互作用；利用生物信息学和基因编辑技术，分析MED23-ncRNA相互作用的结构基础，解析Mediator/ncRNA复合物在转录及表观遗传中的调控作用，从而揭示Mediator及ncRNA的崭新功能和机理。

非编码RNA在转录调节过程发挥非常重要的功能。然而，其调控转录的机制不甚明确。转录中介体作为连接转录因子和通用转录机器的桥梁，发挥着复杂的转录调控功能。转录中介体所处的复杂RNA环境预示着非编码RNA可能与转录中介体相互作用而进一步调控转录的发生。我们通过新的Crosslinking and immunoprecipitation and Sequencing (CLIP)方法发现转录中介体亚基MED23直接结合334种非编码RNA，其中主要是长非编码RNA。进一步地，通过生物信息学的分析方法，我们发现MED23结合RNA的motif偏好性；通过联用CLIP与LC-MS/MS质谱分析的方法，我们检测到MED23结合RNA的氨基酸位点。MED23与非编码RNA的相互作用影响邻近基因的转录水平、调节内含子来源的snoRNA的生成以及假基因的转录。它们的相互作用调节化学突触信号传导、G蛋白耦连信号转导和细胞粘附等生物学过程