TARBP2的类泛素化修饰调控小RNA干扰效率的机制

发布时间：2018-07-29 08:38

【摘要】：非编码小RNA介导基因沉默是调节基因表达的基本却又复杂的分子机制之一。关于如何确保mi RNA/si RNA介导基因沉默有效进行是非编码RNA领域中的未知问题。哺乳动物中,成熟mi RNA通过两步剪切被加工出来。首先,primi RNA在核内被剪切加工复合体Drosha-DGCR8剪切成pre-mi RNA。pre-mi RNA被Ran GTP-Exportin5转运到细胞质中,然后被Dicer-TARBP2复合物识别并剪切加工成双链mi RNA。mi RNA进入RISC(RNA-induced silencing complex,RNA介导沉默复合物)核心组成蛋白Ago2中发挥基因沉默功能。越来越多的证据表明RISC的组装包含两个关键步骤,即双链mi RNA进入RISC和双链mi RNA的解旋。后一步骤中,Ago2能识别并结合mi RNA的有意义链,形成RISC功能中心进一步发挥基因沉默功能,无意义链则被降解,而前一步骤中涉及的具体机制还有待阐明。蛋白质翻译后修饰是生命活动中非常严格的调控层次之一。在小RNA途径中,RNA结合蛋白修饰异常会导致mi RNA失调。相当数量的研究表明,mi RNA失调会引发癌症在内的诸多人类疾病。RNA干扰途径中的关键蛋白修饰在癌症的发生中发挥的作用还并不为人们所知。在第一部分研究中,我们证明了TARBP2的第52位赖氨酸在体内与体外均能发生SUMO化修饰。外周信号刺激,如生长信号刺激能激活MAPK/Erk介导的TARBP2磷酸化修饰并上调其SUMO化修饰,而TARBP2的SUMO化修饰又能抑制其K48连接型泛素化修饰,进而稳定蛋白质。我们还发现,在一些外界因素,如双氧水、低氧等因素刺激下,TARBP2的SUMO化修饰程度会受到调控。在第二部分的研究中,我们发现TARBP2的SUMO化修饰并不影响整体mi RNA的表达,但是能调节小RNA介导基因沉默的效率。从机制上来讲,SUMO化修饰的TARBP2与Dicer一起招募了更多的Ago2来组装成RLC(RISC-loading complex,RISC装载复合物),这种促进作用主要是通过TARBP2上共价连接的SUMO分子与Ago2的SIMs直接相互作用造成的。同时,SUMO化修饰的TARBP2促进了更多的mi RNA/si RNA前体进入RLC,使得Ago2更加稳定,也使得与Dicer-TARBP2结合的mi RNA/si RNA被有效地传递到Ago2中。因此,Ago2能更有效地筛选出mi RNA的有意义链并与之结合,形成有功能的RISC调控靶基因的表达。另外,我们还发现TARBP2的SUMO化修饰对于抑制肿瘤的生长与肿瘤细胞的迁移具有重要作用。本研究首次证明TARBP2能发生SUMO1介导的蛋白质修饰,而且这一修饰能通过增加TARBP2与Ago2和mi RNA/si RNA前体的结合而调控小RNA介导的基因沉默效率。基于本研究取得的实验数据,我们建立了一种蛋白质翻译后修饰调控小RNA干扰效应发挥的细节模型,能帮助我们更好的理解小RNA介导基因沉默机制。我们的研究也为阐明恶性肿瘤的发生机制提供了新的思路。
[Abstract]:Non-coding small RNA mediated gene silencing is one of the basic but complex molecular mechanisms to regulate gene expression. How to ensure mi RNA/si RNA mediated gene silencing is an unknown problem in the field of non-coding RNA. In mammals, mature mi RNA is processed by two-step shearing. In the first place, the primi RNA was shearing in the nucleus by the shear processing complex Drosha-DGCR8, and then the pre-mi RNA.pre-mi RNA was transported into the cytoplasm by Ran GTP-Exportin5. Then the Dicer-TARBP2 complex was recognized and shredded to form a double-stranded mi RNA.mi RNA into the core protein Ago2 (RNA-induced silencing complexRNA-mediated silencing complex), which functions as gene silencing. There is growing evidence that the assembly of RISC involves two key steps: the entry of double-stranded mi RNA into RISC and the unwinding of double-stranded mi RNA. In the latter step, Ago2 can recognize and combine the meaningful chain of mi RNA, and form the functional center of RISC to further exert the function of gene silencing, while the meaningless chain is degraded, but the mechanism involved in the previous step remains to be clarified. Post-translational modification of proteins is one of the very strict regulatory levels in life activities. Aberrant modification of RNA-binding proteins in small RNA pathways leads to misalignment of mi RNA. A considerable number of studies have shown that the role of key protein modifications in the RNAi pathway in many human diseases, including cancer, is unknown. In the first part of the study, we demonstrated that the 52 th position lysine of TARBP2 can be modified by SUMO in vivo and in vitro. Peripheral signal stimulation, such as growth signal stimulation, can activate MAPK/Erk mediated TARBP2 phosphorylation modification and up-regulate its SUMO modification, while TARBP2 SUMO modification can inhibit its K48 ligand ubiquitin modification and stabilize the protein. We also found that the degree of SUMO modification of TARBP2 was regulated by some external factors such as hydrogen peroxide and hypoxia. In the second part of the study, we found that the SUMO modification of TARBP2 did not affect the overall expression of mi RNA, but could regulate the efficiency of gene silencing mediated by small RNA. In mechanism, SUMO-modified TARBP2, together with Dicer, recruited more Ago2 to assemble RLC (RISC-loading complexRISC loading complex), which is mainly caused by the direct interaction between covalently linked SUMO molecules on TARBP2 and Ago2 SIMs. At the same time, the SUMO-modified TARBP2 promoted more mi RNA/si RNA precursors into RLC, made Ago2 more stable, and effectively transferred mi RNA/si RNA combined with Dicer-TARBP2 into Ago2. Therefore, Ago2 can more effectively screen out the meaningful chain of mi RNA and combine with it to form a functional RISC regulatory target gene expression. In addition, we also found that SUMO modification of TARBP2 plays an important role in inhibiting tumor growth and tumor cell migration. This study demonstrated for the first time that TARBP2 can induce SUMO1 mediated protein modification, and this modification can regulate the gene silencing efficiency mediated by small RNA by increasing the binding of TARBP2 to Ago2 and mi RNA/si RNA precursors. Based on the experimental data obtained in this study, we have developed a detailed model for the regulation of small RNA interference by post-translational modification of proteins, which can help us to better understand the mechanism of gene silencing mediated by small RNA. Our research also provides a new idea for elucidating the mechanism of malignant tumor.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q78;R3416

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