多组学整合分析揭示拟南芥Argonaute介导内含子移除的分子机制及相关生物学功能的实验研究

动物中最新研究证据显示Argonaute（AGO）蛋白有调控RNA剪接的重要作用；而植物中鲜有报道。前期，我们提出植物AGO参与内含子移除的假设模型；并已开展拟南芥ago1、ago4突变体的RNA-seq实验，获得了一批基因，其特定内含子的移除依赖AGO1或AGO4活性。基于已完成的AGO1-RIP-seq，我们着重分析在ago1突变体中发生内含子滞留的转录本，发现某些滞留内含子上存在AGO1结合峰，预示AGO1可能通过与RNA的直接结合参与内含子移除。后续，我们将基于IP-LC-MS、RNA结构测序、染色质修饰分析等多组学数据，多维度（AGO—RNA互作、AGO—蛋白互作、RNA二级结构、DNA和组蛋白修饰）解析AGO介导的内含子移除的分子机制。基于拟南芥转基因等实验，针对特定基因的剪接异构体，研究依赖AGO活性的内含子移除的生物学意义。上述研究将增进人们对植物AGO蛋白功能的全面理解。

Recently, increasing pieces of evidence in animals pointed to the functional importance of the Argonaute (AGO) protein in RNA splicing regulation. However, rare reports were found for plants. Our group has proposed a possible mechanistic model for AGO-mediated intron removal in plants. RNA-seq analysis of the ago1 and ago4 mutants of Arabidopsis thaliana enabled us to identify a set of genes whose intron removal specifically relied on the activity of the AGO1 or AGO4 protein. Besides, based on the AGO1-RIP-seq analysis, the AGO1 binding peaks were found within some of the intronic regions whose retention was observed in the ago1 mutant. It indicates the possibility that the AGO1 protein might act on the process of intron removal through direct binding of the intronic sequences. For the future plan, we will carry out integrated analysis of multiple omics datasets (such as IP-LC-MS, RNA structurome, and chromatin modifications), in order to uncover the molecular mechanism of the AGO-mediated intron removal from multiple dimensions (i.e. AGO—RNA interactions, AGO—protein interactions, RNA secondary structures, DNA methylation and histone modifications). Based on transgenic and other molecular studies on specific alternative splicing isoforms in Arabidopsis, we aim to reveal the biological roles of the AGO-dependent intron removal event. Taken together, the expected results of the above project will expand the current knowledge on the functions of the AGO proteins in plants.