酵母RNA外切体复合物的冷冻电镜结构分析

发布时间：2018-07-01 16:10

本文选题：外切体 + Rrp44　；参考：《清华大学》2016年博士论文

【摘要】：真核生物RNA外切体是多亚基蛋白复合物,由九亚基核心和Rrp44亚基组成。复合物中,九亚基核心无核苷酸酶活性,而Rrp44兼具从3’-5’的核苷酸外切酶和内切酶活性。大量功能研究表明,在细胞核和细胞质内众多辅助因子的协助下,外切体参与信使RNA,核糖体RNA,转运RNA,小RNA以及大量长非编码RNA的加工代谢,且影响重大。通过电镜单颗粒及生化实验分析,我们揭示了酵母外切体内应对不同RNA底物的多条降解通路。在过核降解通路中,单链RNA将穿过外切体的核心孔道进入Rrp44亚基被加工处理,同时诱发Rrp44亚基的构象变化。在直接降解通路中,RNA可以避开核心孔道而直接结合到Rrp44亚基的外切酶活性中心,且不诱发Rrp44的构象变化。同时,我们利用电镜技术对RNA在外切体复合物中降解的过程进行了统计分析和重现,为外切体复合物对不同类型的RNA底物的降解模式提供了机理上的解释。与此同时,通过对十亚基外切体复合物内源纯化方法的细节调整,我们成功地纯化并获得了酵母内源表达的外切体与其细胞质辅助因子Ski7的复合物样品,并利用冷冻单颗粒三维重构获得了该复合物的多种状态下的高分辨率结构。其中,无底物状态的重构模型分辨率为4.2埃,底物结合状态重构模型的分辨率为5.8埃,我们因而可以从原子水平来分析两种状态的结构差异。结合外切体与其它种类RNA复合物的结构解析,我们大致揭示了RNA经由核心孔道诱发Rrp44构象变化以及激活降解进程的分子机制。结构的分析亦阐明了Ski7与外切体相互作用的方式,与已解析的外切体和Rrp6复合物结构对比可知,Ski7的N端结构域与Rrp6的C端结构域在二级结构排布上极其相似,且与外切体相互作用模式也基本一致,这在一定程度上解释了细胞内不同外切体族群如何差异性地在多种RNA加工处理事件中独立工作。
[Abstract]:RNA exon of eukaryote is a multisubunit protein complex composed of 9 subunit core and RRP 44 subunit. In the complex, nucleotidase activity was not found in the core of the nine subunits, while RRP 44 had both exonuclease and endonuclease activity from 3 to 5 'nucleotides. A large number of functional studies have shown that exosomes are involved in the processing and metabolism of messenger RNAs, ribosomal RNAs, transporter RNAs, small RNAs and a large number of long non-coding RNAs, with the help of many auxiliary factors in the nucleus and cytoplasm. By means of electron microscope single particle and biochemical experiments, we have revealed many pathways of degradation of different RNA substrates in yeast. In the transnuclear degradation pathway, single strand RNA passes through the core pore of the exoskeleton into the Rrp44 subunit to be processed, and at the same time induces the conformation change of the Rrp44 subunit. In the direct degradation pathway, the RNA could avoid the core pore and bind directly to the exonuclease active center of Rrp44 subunit, and did not induce the conformation change of Rrp44. At the same time, we used electron microscopy to analyze and reproduce the degradation process of RNA in exonuclear complexes, which provided a mechanism for the degradation of different types of RNA substrates by exoskeleton complexes. At the same time, by adjusting the details of the method of purification of the exosome complex, we have successfully purified and obtained the complex sample of the exon expressed by yeast and its cytoplasmic cofactor Ski7. The high resolution structure of the composite was obtained by three-dimensional reconstruction of single-particle cryopreservation. The resolution of the reconstruction model without substrate state is 4.2 and the resolution of the substrate combined state reconstruction model is 5.8 A. therefore, we can analyze the structural differences of the two states from the atomic level. In combination with the structural analysis of the exosomes and other RNA complexes, we have revealed the molecular mechanism of RNA inducing the conformation change of RRP 44 through the core channels and activating the degradation process. The structure analysis also illustrates the interaction between Ski7 and the exoskeleton. The comparison of the structure of Ski7 and Rrp6 complex shows that the N-terminal domain of Ski7 and the C-terminal domain of Rrp6 are very similar in the arrangement of secondary structures. And the pattern of interaction with exonuclease is basically consistent, which to some extent explains how different exonuclease populations in cells work independently in various RNA processing events.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q75

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