Argonaute (AGO)蛋白MEL1的磷酸化修饰对减数分裂的调控机制研究

In eukaryotes, AGOs bind to small RNA (sRNA) and other biomacromolecules to form RNA-induced silencing complexs (RISCs), which is involved in RNA-mediated DNA methylation (RdDM), and mRNA cleavage or degradation. Phosphorylation of AGO can alter its own structure, which will affect its association with sRNA and other proteins, and regulate the dynamic balance of AGO protein in different subcellular structures. It is worth noting that the rice AGO protein, MEL1, is specifically expressed in germ cells, and loss function of MEL1 causes pollen mother cells (PMC) to stop in the early stages of meiosis and affects the modification of histone H3, including H3K9me2, throughout the whole genome, and leads to complete infertility in rice. we found that MEL1 binds to the ribosomal protein RPS18 and H3K9me2 in the early stage of meiosis. Short-cut of MEL1 protein and dephosphorylation of MEL1 affect its binding to histone H3. Here we will construct phosphorylation site-directed mutations on different domains of MEL1 and analyze the affection of MEL1 phosphorylation on its subcellular localization and the physical interaction with other proteins, to reveal the regulatory mechanism of MEL1-RISC constituted by different molecules in each subcellular structure. On the other hand, the effects of MEL1 phosphorylation on the PMC development and genomic DNA methylation during meiosis will be studied to understand the gene expression and epigenetic modification during meiosis, which will provide a theoretical basis for genetic breeding.

真核生物的AGO蛋白与sRNA及其他生物大分子结合形成RNA诱导的沉默复合物(RISC)，参与sRNA介导的DNA甲基化和mRNA的剪切或降解，调控生物体生长发育的几乎所有的生命过程。磷酸化修饰可以改变AGO蛋白构造，影响其与sRNA和其他蛋白结合。水稻AGO蛋白MEL1缺失导致花粉母细胞停止在减数分裂早期，影响组蛋白H3修饰(包括H3K9me2)，导致败育。我们的近期研究表明MEL1与核糖体蛋白RPS18和H3K9me2结合，且不同结构域缺失和脱磷酸化影响其与H3结合。本课题将构建MEL1磷酸化位点突变体，研究磷酸化位点突变对MEL1亚细胞定位及其与其他蛋白物理互作的影响，揭示磷酸化对MEL1在各个亚细胞结构中与不同蛋白构成不同RISC的调控机制。同时研究MEL1磷酸化对花粉母细胞发育和基因组DNA甲基化的影响，进一步理解减数分裂中基因表达和表观修饰调控机制，为遗传育种提供理论基础。

在项目实施期间，我们对水稻AGO蛋白MEL1的磷酸化修饰对减数分裂的调控机制开展研究。结合我们的阶段性实验结果和他人的实时研究进展，我们对本项目进行了调整。同时，我们对拟南芥和毛竹的AGO蛋白介导的植物生殖发育过程的作用机制进行了扩展研究。首先，我们筛选出MEL1可能的磷酸化位点，发现这些位点突变会影响MEL1与其互作蛋白的结合，从而影响MEL1生物学功能。利用CLIP-seq技术对MEL1蛋白结合的靶RNA进行分析，建立CLIP-seq数据分析流程，并初步鉴定出MEL1调控的靶基因。其次，我们对拟南芥AGO1在植物生殖生长过程的作用机制研究，挖掘到了AGO1介导的植物花期调控和花粉发育过程中的新的作用分子。最后，我们对毛竹AGO基因进行克隆和功能分析，结果表明毛竹的AGO蛋白对植物的生长发育具有重要功能。这些结果表明，AGO基因的研究为作物经济性状改良提供了理论基础。