植物DNA甲基化相关RNA聚合酶的结构与功能研究

植物DNA甲基化的建立依赖于植物独特的非编码RNA指导的DNA甲基化途径——RdDM途径。该途径以两个植物特有的RNA 聚合酶，RNA聚合酶IV和V（Pol IV和V），为核心展开介导DNA甲基化的发生。在通路上游，Pol IV在染色质特定位点转录生成单链RNA；该单链RNA被Pol IV结合的RNA依赖的RNA聚合酶RDR2合成双链前体siRNA；前体siRNA被植物特有的Dicer酶DCL3切割至24/23 nt后其中一条24 nt的成熟siRNA装载入AGO4中。在通路下游，Pol V转录生成长链非编码RNA（lncRNA）后，Pol V-lncRNA复合物作为支架与AGO4上24 nt siRNA结合并进一步招募下游DNA甲基化酶DRM2介导位点特异性DNA甲基化。Pol IV和Pol V都是由经典的Pol II进化而来，但是与Pol II相比它们的生化性质与功能有了较大的变化。研究表明Pol IV与Pol V体外转录活性相较与Pol II更低。转录过程中，Pol IV比Pol II更容易出错，而Pol V具有更高的保真度。在功能上，与Pol II直接合成RNA转录本不同，Pol V自身主要作为支架来完成染色质定位功能。这些与Pol II不同的生化性质与功能，目前尚未有很好的阐释。

本研究针对Pol V的转录和调控，开展了Pol V的结构与功能研究。利用冷冻电镜技术，解析了Pol V单独的结构及其处于转录延伸状态的复合物结构。通过对其结构的分析，发现Pol V与Pol II在结构上有显著的不同之处：Pol V的第二亚基NRPE2的第495位酪氨酸的残基会插入到DNA双链打开的位置；NRPE2跟非模板链紧密结合。生化实验显示Pol V在遇到双链DNA时，转录的速率出现了明显的减缓，而Pol II并没有此类现象。由此推测是由于NRPE2上第495位酪氨酸的存在，使得DNA双链打开这一过程被阻滞，从而导致Pol V的转录速率低于Pol II。另一方面，NRPE2与非模板链的紧密结合，增强了Pol V回溯RNA的能力，使得Pol V的外切酶活性大大提高，这促使Pol V拥有了比Pol II更高的保真度。整合起来，Pol V比Pol II更慢的转录速率，及其与转录泡更加紧密的结合，导致Pol V可以更长时间的滞留在染色质，这也就解释了Pol V通过转录阻滞和增强回溯等机制延长染色质滞留而高效介导DNA甲基化的原因。

通过对Pol V结构与生化性质的研究，本研究进一步推导出与Pol V含有同样NRPE2亚基的Pol IV的部分生化性质与Pol V相同。即：与Pol II相比，Pol IV的转录速率较低；其同样依靠第二亚基对非模板链的结合获得了更强的回溯能力。基于此，通过对Pol IV-RDR2的结构进一步分析，发现Pol IV-RDR2之间形成的RNA通道长度约20 nt，加上10 nt的转录泡长度，形成了一个共计30 nt长度的RNA通道。长度超过30 nt的Pol IV转录的RNA，包含10 nt的转录泡及20 nt的聚合酶间通道，会触发非模板链DNA的强向后回溯，有效地将Pol IV RNA的3′端传递给RDR2，从而启动双链RNA的合成。而长度小于30 nt的Pol IV RNA虽然可以进入聚合酶间通道，但由于没有足够长度的RNA来维持转录泡以供NRPD(E)2结合，因此导致回溯减弱，从而无法高效将Pol IV RNA转移给RDR2，造成dsRNA合成效率较低。这样的双重测量机制，完整的解释了植物中Pol IV的转录本长度主要分布在26-45 nt范围内，且峰值为30 nt的现象。

综上所述，本研究系统解析了植物特有的Pol V的结构和生化性质：通过对结构的分析，结合生化实验，解释了Pol V转录速率低于Pol II的原因及其转录后滞留在染色质上发挥染色质定位作用的分子机制。进一步根据Pol IV和Pol V在结构和生化相似之处，提出了一种由Pol IV-RDR2介导的“siRNA前体生成的双重测量模型”，Pol IV-RDR2测量模型的提出，解释了siRNA前体长度主要分布在26-45 nt长度的分子机制。这些工作成功诠释了RdDM通路中非编码RNA的生物合成机制，为全面解析RdDM的分子原理起到了重要的推动作用。

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