参与ABA和渗透胁迫应答的mRNA降解途径研究

The differential transcription of genes determines the diversity of eukaryotic cell, as well as the adaption of eukaryotic cells to environmental changes. Unlike well-studied epigenetic regulation, initiation of transcription and mRNA splicing, the regulation of mRNA decay in plant is still largely unknown. In our previous studies, we found that a key regulator in mRNA decay, PAT1, is involved in Abscisic Acid (ABA) signaling and salt stress responses. The pat1 mutant plants are hypersensitive to ABA and NaCl at germination and early growth stages. SnRK2 kinases, the core components in ABA signaling and osmotic stress responses, could phosphorylate PAT1 in vitro. Based these results, we proposed that PAT1 mediated mRNA decay plays critical roles in transcriptional regulation downstream of SnRK2s. We’ll identify the phosphorylation sites of PAT1 and study the mechanism underlying SnRK2s regulated PAT1 functions in planta. We’ll also identify the role of mRNA decay in transcription of ABA and salt stress responsive genes by isolating the target mRNA of PAT1. The PAT1-interacting proteins that function in the recruitment and activation of decapping complex will also be isolated and studied. These studies will help us to understand the mRNA decay processes in plant and reveal its role in plant abiotic stress signaling.

真核基因转录的差异决定细胞的不同功能和个体对不同环境的适应。相对于表观遗传调控、转录起始和mRNA剪切等调节过程，目前对于植物mRNA稳定性的调控仍了解较少。我们发现mRNA降解途径中的关键蛋白PAT1参与了植物激素脱落酸和渗透胁迫的应答。pat1缺失突变体对ABA和NaCl处理敏感。ABA和渗透胁迫途径的核心组分SnRK2蛋白激酶能磷酸化PAT1蛋白。本项目拟进一步研究SnRK2对PAT1蛋白的磷酸化，鉴定PAT1蛋白的磷酸化位点，分析SnRK2磷酸化调控PAT1功能的分子机制。鉴定PAT1直接调控的下游靶基因，解析PAT1影响ABA和盐胁迫应答基因转录的细节。分离PAT1相互作用蛋白，鉴定mRNA降解过程中介导PAT1招募和激活5’去帽复合体的中间蛋白，分析PAT1调控mRNA降解的分子机制。这一研究有助于深入理解植物mRNA降解的调控过程，并发现植物应答非生物胁迫的新途径。

真核基因转录的差异决定细胞的不同功能和个体对不同环境的适应。相对于表观遗传调控、转录起始和mRNA剪切等调节过程，目前对于植物mRNA稳定性的调控仍了解较少。利用ABA和渗透胁迫信号通路重要元件SnRK2蛋白质磷酸化组学寻找参与ABA和渗透胁迫的下游蛋白，我们发现mRNA降解途径中的关键蛋白PAT1可能被SnRK2磷酸化。体外pull down实验结果显示SnRK2与PAT1可以直接相互作用，split-luc实验结果显示SnRK2与PAT1在体内可以相互作用。而且体外磷酸化显示SnRK2可以磷酸化PAT1的S83, S171, S393位点。PAT1的突变体pat1对ABA和NaCl更敏感，并且NaCl处理可以增加PAT1的蛋白含量。RNAseq结果显示NaCl处理可导致2296个基因上调，其中150个基因是依赖于PAT1的，2393个下调基因中407个基因依赖于PAT1，其中包括ABA2,NCED3等参与ABA合成的基因，PAT1可能直接介导了这些基因的降解。这一研究有助于深入理解植物mRNA降解的调控过程。