本文選題：外切體 + Rrp44��； 參考：《清華大學(xué)》2016年博士論文

【摘要】：真核生物RNA外切體是多亞基蛋白復(fù)合物,由九亞基核心和Rrp44亞基組成。復(fù)合物中,九亞基核心無核苷酸酶活性,而Rrp44兼具從3’-5’的核苷酸外切酶和內(nèi)切酶活性。大量功能研究表明,在細胞核和細胞質(zhì)內(nèi)眾多輔助因子的協(xié)助下,外切體參與信使RNA,核糖體RNA,轉(zhuǎn)運RNA,小RNA以及大量長非編碼RNA的加工代謝,且影響重大。通過電鏡單顆粒及生化實驗分析,我們揭示了酵母外切體內(nèi)應(yīng)對不同RNA底物的多條降解通路。在過核降解通路中,單鏈RNA將穿過外切體的核心孔道進入Rrp44亞基被加工處理,同時誘發(fā)Rrp44亞基的構(gòu)象變化。在直接降解通路中,RNA可以避開核心孔道而直接結(jié)合到Rrp44亞基的外切酶活性中心,且不誘發(fā)Rrp44的構(gòu)象變化。同時,我們利用電鏡技術(shù)對RNA在外切體復(fù)合物中降解的過程進行了統(tǒng)計分析和重現(xiàn),為外切體復(fù)合物對不同類型的RNA底物的降解模式提供了機理上的解釋。與此同時,通過對十亞基外切體復(fù)合物內(nèi)源純化方法的細節(jié)調(diào)整,我們成功地純化并獲得了酵母內(nèi)源表達的外切體與其細胞質(zhì)輔助因子Ski7的復(fù)合物樣品,并利用冷凍單顆粒三維重構(gòu)獲得了該復(fù)合物的多種狀態(tài)下的高分辨率結(jié)構(gòu)。其中,無底物狀態(tài)的重構(gòu)模型分辨率為4.2埃,底物結(jié)合狀態(tài)重構(gòu)模型的分辨率為5.8埃,我們因而可以從原子水平來分析兩種狀態(tài)的結(jié)構(gòu)差異。結(jié)合外切體與其它種類RNA復(fù)合物的結(jié)構(gòu)解析,我們大致揭示了RNA經(jīng)由核心孔道誘發(fā)Rrp44構(gòu)象變化以及激活降解進程的分子機制。結(jié)構(gòu)的分析亦闡明了Ski7與外切體相互作用的方式,與已解析的外切體和Rrp6復(fù)合物結(jié)構(gòu)對比可知,Ski7的N端結(jié)構(gòu)域與Rrp6的C端結(jié)構(gòu)域在二級結(jié)構(gòu)排布上極其相似,且與外切體相互作用模式也基本一致,這在一定程度上解釋了細胞內(nèi)不同外切體族群如何差異性地在多種RNA加工處理事件中獨立工作。
[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.
【學(xué)位授予單位】：清華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q75

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本文編號：2088329