拟南芥RdDM通路新因子IYO与QQT2的鉴定及其作用机制研究

发布时间：2018-05-02 16:08

  本文选题：RdDM + IYO　； 参考：《北京协和医学院》2016年博士论文

【摘要】：DNA甲基化是一种在真核生物中保守的重要表观遗传修饰。植物中的DNA甲基化可以由RNA介导的DNA甲基化(RNA-directed DNA methylation, RdDM)通路来建立。RdDM通路需要小RNA (small RNA, sRNA)的介导以及植物中特有的两种依赖于DNA的RNA聚合酶(DNA-dependent RNA polymerase) Pol Ⅳ和Pol Ⅴ的共同参与。Pol Ⅳ和Pol Ⅴ与经典的RNA聚合酶Pol Ⅱ同源。Pol Ⅳ转录本被依赖于RNA的RNA聚合酶2(RNA dependent RNA polymerase 2, RDR2)和Dicer类似蛋白3(Dicer-like protein 3, DCL3)加工形成24个核苷酸(nucleotide, nt)长度的异染色质相关的小干扰RNA (heterochromatic small interfering RNA, hc-siRNA),在细胞质中与ARGONAUTE4 (AGO4)蛋白组装形成RNA沉默复合物(RNA-Induced Silencing Complex, RISC)。该沉默复合物随后被转运进入细胞核内,由siRNA靶向介导其与Pol Ⅴ转录本的结合,并招募DNA甲基转移酶2(Domains Rearranged Methyltransferase 2, DRM2)建立与hc-siRNA互补DNA的甲基化修饰。为了发现参与RdDM通路的新因子,我们利用拟南芥AG04自身启动子驱动表达的融合绿色荧光蛋白(Green fluorescent protein, GFP)的AG04转基因系,建立了有效针对RdDM通路的正向遗传学筛选体系。当RdDM通路发生缺陷导致hc-siRNA的水平降低时,AG04蛋白的稳定性下降,从而转基因系中GFP-AGO4的荧光信号将相应减弱。通过诱变筛选,我们从该体系中获得了68个AG04蛋白稳定性下降(defective in AGO4 stability, das)的突变体,其中包括65个RdDM通路已知组分的突变体和3个新突变体。本研究中,我们主要对dasl和das2两个新突变体进行分析。在dasl和das2突变体中,24-nt hc-siRNA的积累以及RdDM靶标位点的DNA甲基化在全基因组水平大幅下降,说明DAS1和DAS2是RdDM通路的必需组分。我们进一步分析发现,dasl和das2突变体中受影响的位点与Pol Ⅳ和Pol Ⅴ的最大亚基突变体nrpd1和nrpe1所影响的位点几乎完全重叠,暗示DAS1和DAS2可能是通过调控Pol Ⅳ和Pol Ⅴ的功能而参与RdDM的。图位克隆显示,DAS1和DAS2分别编码MINIYO(IYO)和QUATRE-QUART2 (QQT2),它们在动物和酵母中的同源物均被发现为PolII复合物的附属组分,且QQT2在酵母中的同源物Npa3被认为作为分子伴侣参与Pol Ⅱ的组装。通过生化实验,我们发现IYO和QQT2在拟南芥细胞中能够与RNA聚合酶的多个亚基相互作用,包括Pol Ⅱ、Pol Ⅳ和Pol Ⅴ共有的第三、第十和第十一亚基。通过进一步研究我们发现,das1/iyo和das2/qqt2突变体中,Pol Ⅴ全酶复合物并不完整,核心催化亚基NRPE1与其它亚基之间的结合程度显著下降,说明IYO和QQT2参与Pol Ⅴ的组装。与此相一致,我们发现das1/iyo和das2/qqt2突变体中NRPE1蛋白的积累量明显降低,NRPE1在细胞核中的分布减少,进一步说明IYO和QQT2的功能缺失导致Pol Ⅴ不能组装,进而使得核心催化亚基NRPE1易于降解且不能顺利入核。本研究鉴定了RdDM通路的两个全新因子IYO和QQT2,并证明它们通过调节Pol Ⅴ的组装而参与RdDM通路,揭示了RdDM通路中新的调控层面。此外,Pol Ⅴ与Pol Ⅱ、Pol Ⅳ在组成上高度相似,它们共用大部分亚基,未来进一步阐明植物中IYO和QQT2参与Pol Ⅴ组装的机制,将有助于我们认识真核生物中其它RNA聚合酶的组装过程。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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1 李瑶曦;拟南芥RdDM通路新因子IYO与QQT2的鉴定及其作用机制研究[D];北京协和医学院;2016年

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