本文選題：RdDM + IYO��； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：DNA甲基化是一種在真核生物中保守的重要表觀遺傳修飾。植物中的DNA甲基化可以由RNA介導(dǎo)的DNA甲基化(RNA-directed DNA methylation, RdDM)通路來建立。RdDM通路需要小RNA (small RNA, sRNA)的介導(dǎo)以及植物中特有的兩種依賴于DNA的RNA聚合酶(DNA-dependent RNA polymerase) Pol Ⅳ和Pol Ⅴ的共同參與。Pol Ⅳ和Pol Ⅴ與經(jīng)典的RNA聚合酶Pol Ⅱ同源。Pol Ⅳ轉(zhuǎn)錄本被依賴于RNA的RNA聚合酶2(RNA dependent RNA polymerase 2, RDR2)和Dicer類似蛋白3(Dicer-like protein 3, DCL3)加工形成24個核苷酸(nucleotide, nt)長度的異染色質(zhì)相關(guān)的小干擾RNA (heterochromatic small interfering RNA, hc-siRNA),在細(xì)胞質(zhì)中與ARGONAUTE4 (AGO4)蛋白組裝形成RNA沉默復(fù)合物(RNA-Induced Silencing Complex, RISC)。該沉默復(fù)合物隨后被轉(zhuǎn)運進(jìn)入細(xì)胞核內(nèi),由siRNA靶向介導(dǎo)其與Pol Ⅴ轉(zhuǎn)錄本的結(jié)合,并招募DNA甲基轉(zhuǎn)移酶2(Domains Rearranged Methyltransferase 2, DRM2)建立與hc-siRNA互補(bǔ)DNA的甲基化修飾。為了發(fā)現(xiàn)參與RdDM通路的新因子,我們利用擬南芥AG04自身啟動子驅(qū)動表達(dá)的融合綠色熒光蛋白(Green fluorescent protein, GFP)的AG04轉(zhuǎn)基因系,建立了有效針對RdDM通路的正向遺傳學(xué)篩選體系。當(dāng)RdDM通路發(fā)生缺陷導(dǎo)致hc-siRNA的水平降低時,AG04蛋白的穩(wěn)定性下降,從而轉(zhuǎn)基因系中GFP-AGO4的熒光信號將相應(yīng)減弱。通過誘變篩選,我們從該體系中獲得了68個AG04蛋白穩(wěn)定性下降(defective in AGO4 stability, das)的突變體,其中包括65個RdDM通路已知組分的突變體和3個新突變體。本研究中,我們主要對dasl和das2兩個新突變體進(jìn)行分析。在dasl和das2突變體中,24-nt hc-siRNA的積累以及RdDM靶標(biāo)位點的DNA甲基化在全基因組水平大幅下降,說明DAS1和DAS2是RdDM通路的必需組分。我們進(jìn)一步分析發(fā)現(xiàn),dasl和das2突變體中受影響的位點與Pol Ⅳ和Pol Ⅴ的最大亞基突變體nrpd1和nrpe1所影響的位點幾乎完全重疊,暗示DAS1和DAS2可能是通過調(diào)控Pol Ⅳ和Pol Ⅴ的功能而參與RdDM的。圖位克隆顯示,DAS1和DAS2分別編碼MINIYO(IYO)和QUATRE-QUART2 (QQT2),它們在動物和酵母中的同源物均被發(fā)現(xiàn)為PolII復(fù)合物的附屬組分,且QQT2在酵母中的同源物Npa3被認(rèn)為作為分子伴侶參與Pol Ⅱ的組裝。通過生化實驗,我們發(fā)現(xiàn)IYO和QQT2在擬南芥細(xì)胞中能夠與RNA聚合酶的多個亞基相互作用,包括Pol Ⅱ、Pol Ⅳ和Pol Ⅴ共有的第三、第十和第十一亞基。通過進(jìn)一步研究我們發(fā)現(xiàn),das1/iyo和das2/qqt2突變體中,Pol Ⅴ全酶復(fù)合物并不完整,核心催化亞基NRPE1與其它亞基之間的結(jié)合程度顯著下降,說明IYO和QQT2參與Pol Ⅴ的組裝。與此相一致,我們發(fā)現(xiàn)das1/iyo和das2/qqt2突變體中NRPE1蛋白的積累量明顯降低,NRPE1在細(xì)胞核中的分布減少,進(jìn)一步說明IYO和QQT2的功能缺失導(dǎo)致Pol Ⅴ不能組裝,進(jìn)而使得核心催化亞基NRPE1易于降解且不能順利入核。本研究鑒定了RdDM通路的兩個全新因子IYO和QQT2,并證明它們通過調(diào)節(jié)Pol Ⅴ的組裝而參與RdDM通路,揭示了RdDM通路中新的調(diào)控層面。此外,Pol Ⅴ與Pol Ⅱ、Pol Ⅳ在組成上高度相似,它們共用大部分亞基,未來進(jìn)一步闡明植物中IYO和QQT2參與Pol Ⅴ組裝的機(jī)制,將有助于我們認(rèn)識真核生物中其它RNA聚合酶的組裝過程。
[Abstract]:DNA methylation is an important epigenetic modification that is conserved in eukaryotes. DNA methylation in plants can be based on the RNA mediated DNA methylation (RNA-directed DNA methylation, RdDM) pathway to establish the.RdDM pathway that requires small RNA (small RNA), as well as the two dependent polymerase in plants. ENT RNA polymerase) Pol IV and Pol V are involved in.Pol IV and Pol V and the classical RNA polymerase Pol II homologous.Pol IV transcript is dependent on RNA RNA polymerase 2 and similar protein 3 to form 24 nucleotides. The small interference RNA (heterochromatic small interfering RNA, hc-siRNA) is assembled in the cytoplasm with the ARGONAUTE4 (AGO4) protein to form a RNA silencing complex (RNA-Induced Silencing Complex,). The silenced complex is then transported into the nucleus. DNA methyltransferase 2 (Domains Rearranged Methyltransferase 2, DRM2) was used to establish methylation modification of hc-siRNA complementary DNA. In order to find a new factor to participate in the RdDM pathway, we have established the transgenic lines of the fused green fluorescent protein (Green fluorescent protein), which are expressed in the AG04 self promoter of Arabidopsis. A positive genetic screening system for the RdDM pathway. When the RdDM pathway is defective and the level of hc-siRNA decreases, the stability of the AG04 protein decreases, and the fluorescence signal of the GFP-AGO4 in the transgenic line will be reduced accordingly. Through the mutation screening, we obtained 68 AG04 protein stability drops (defective in AGO4 stab) from this system. Mutants of ility, DAS), including 65 known components of RdDM pathway and 3 new mutants. In this study, we mainly analyzed two new mutants of DASL and das2. In DASL and das2 mutants, the accumulation of 24-nt hc-siRNA and DNA methylation of RdDM target loci decreased significantly at the whole genome level, indicating DAS1 and DAS2 is an essential component of the RdDM pathway. We further found that the affected loci in the DASL and das2 mutants are almost completely overlapped with the loci affected by the largest subunit mutants of Pol IV and Pol V, nrpd1 and nrpe1, suggesting that DAS1 and DAS2 may be involved in the regulation of Pol IV and Pol V. 1 and DAS2, respectively, encode MINIYO (IYO) and QUATRE-QUART2 (QQT2), and their homologues in animals and yeast are found to be subsidiary components of the PolII complex, and the homologous Npa3 in the yeast is considered as a molecular chaperone involved in the assembly of Pol II. Through biochemical experiments, we found that IYO and QQT2 can be associated with RNA in Arabidopsis cells. The multiple subunits of the polymerase interact, including the third, tenth and eleventh subunits of Pol II, Pol IV and Pol v. Through further studies, we found that the Pol V total enzyme complex was not complete in the das1/iyo and das2/qqt2 mutants, and the binding degree between the core subunit NRPE1 and other subunits decreased significantly, indicating IYO and QQT2. In accordance with this, we found that the accumulation of NRPE1 protein in the das1/iyo and das2/qqt2 mutants decreased significantly, and the distribution of NRPE1 in the nucleus decreased, further indicating that the functional deletion of IYO and QQT2 resulted in the failure of Pol V to be assembled and that the core catalytic subunit NRPE1 was easily degraded and failed to enter the nucleus smoothly. Two new factors, IYO and QQT2, of the RdDM pathway were identified, and they were shown to participate in the RdDM pathway by regulating the assembly of Pol V, revealing the new regulatory level in the RdDM pathway. In addition, Pol V and Pol II, Pol IV are highly similar in composition. They share most subunits and further clarify that IYO and QQT2 in plants are involved in Pol V assembly in the future. The mechanism will help us to understand the assembly process of other RNA polymerase in eukaryotes.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

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1 李瑤曦;擬南芥RdDM通路新因子IYO與QQT2的鑒定及其作用機(jī)制研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

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本文編號：1834587