RNA沉默通路新成员在植物与病毒互作中的功能研究

RNA silencing plays key roles in plant resistance to the invasion of virus and other genetic material. However, we know little about the biogenesis and function of RNA silencing at cell biology level, which restricts our understanding and apply of RNA silencing. AGOs are the catalytic proteins in RNA silencing, of which AGO2 has special roles in antiviral immunity. Our preliminary data had shown that: RNA binding protein 1 (RBP1) was found to be a novel component of AGO2 complex, RBP1 and its similar protein RBP2 probable localized in RNA-protein granules both in cytoplasm and nuclear; the malfunction of RBP1 and RBP2 increased plant resistance to viruses, respectively; the subcellular localization of RBPs were changed upon the infection of viruses. Next, we will study the biogenesis and function of RNA silencing at cell biology level. We will focus on the interaction of AGO with RBP1 and RBP2. The co-regulation of RNA-protein granules upon virus infection will also been studied. The fulfillment of this proposal will improve our understanding of the biogenesis and function of RNA silencing at cell biology level, which will finally benefit the control of plant viruses.

RNA沉默对植物抵御外源遗传物质入侵具有重要作用。其分子生物学作用机制已得到深入阐释，然而，其发生和作用所依赖的细胞生物学基础尚有待研究。此方面的研究有助于拓宽人们对RNA沉默的认知和对之更好地利用。申请人前期研究发现：RNA结合蛋白RBP1为AGO2复合体新成员，RBP1与其高同源性蛋白RBP2在细胞质和细胞核中均呈点状分布，很可能定位于RNA-蛋白小体；突变体对病毒抗性增强；病毒入侵改变RBP1/RBP2的亚细胞定位。申请人聚焦RNA沉默发生和作用的细胞生物学基础，将以AGO与RBP1/RBP2的作用为研究主线，将细胞生物学、分子生物学和生物化学研究有机融合，深度解析RNA-蛋白小体与RNA沉默的关系，以及其在抗病毒防御中的协同调控机制。研究结果不仅将揭示RNA沉默新基因的分子功能，也将在细胞学层面完善对RNA沉默发生和作用机制的认知，为有效控制病毒侵染提供科学依据。

RNA沉默是植物抵御病毒入侵的重要防御手段，虽然分子生物学层面的作用机制已得到深入阐释，但其发挥作用所依赖的细胞生物学基础仍有待探究。切割复合体是miRNAs前体加工的功能复合体，在细胞核中呈特殊形态分布，即1-4个无膜点状分布，被称为切割小体（Dicing body）。本项目围绕切割小体的细胞学形成机制，通过研究RNA沉默通路新成员RH6/RH8/RH12（RNA Helicase 6/8/12，为申请书中的RBPs）的功能，挖掘RNA沉默在病毒与植物互作中的调控或被调控，深入认识RNA沉默并对之更好地利用。通过生物化学和细胞生物学等方法，证明了切割小体为液-液相无膜细胞器，而RH6/RH8/RH12作为相变性质蛋白与切割复合体相互作用，促进了切割小体的形成。rh6rh8rh12突变体中切割小体的形成被大大减弱且miRNA前体累积水平下降。病毒与植物互作过程中，一方面，通过劫持RH6/RH8/RH12，在细胞质中形成无膜结构病毒小体，促进病毒的扩繁；另一方面，由于RH6/RH8/RH12被挟持，切割小体的形成受到病毒的抑制。