RNA结合蛋白RZ-1B/1C调控种子萌发的分子机制

The Seeds are of particular importance as they serve not only as an important form of life but also as major food resource of human. Seed germination is an important hall mark during development, through which the plant start their vegetative growth. Seed germination has being extensively studied during the past 20 years. Previous studies have highlighted the importance of plant hormone, transcription factors and post-translational modifications in seed germination control, while little is known about how post-transcriptional mechanism is involved in this process. We previously characterized two Arabidopsis RNA-binding proteins RZ-1B and RZ-1C, which are functionally redundant and play important roles in regulating RNA splicing and general gene expression. Loss of function of both proteins lead to pleiotropic phenotypes including severely delayed germination. Our preliminary data indicate that RZ-1B/1C regulate germination speed through integrating the GA and ABA metabolism. Such integration has been extensively studied but still largely unclear at the moment. Therefore, we propose here to use RZ-1B/1C as an example to study the role of post-transcriptional regulation during seed germination. We will use comprehensive tools to clarify the roles of RZ-1B/1C in the existing network of plant hormones. We will also identify the direct binding targets of RZ-1C through iCLIP and investigate the role of RZ-1C in regulating transcriptional initiation, RNA splicing and RNA decay separately. Furthermore, we will look for signs of potential coordination between these transcriptional and post-transcriptional events as regulated by RZ-1B/1C or as timely regulated during germination. Our work will provide comprehensive knowledge about the role of post-transcriptional gene regulation during seed germination.

种子是高等植物重要的生命形式及人类主要的食物来源。种子萌发作为一个重要发育事件一直被关注，过去的研究揭示了植物激素，转录因子及蛋白翻译后修饰等经典机制在萌发中的重要作用，而对转录后水平调控在其中的作用少有涉及。申请人之前鉴定了两个重要的RNA结合蛋白RZ-1B/1C，发现它们结合染色质并广泛参与了基因表达和RNA剪接的调控。rz-1b rz-1c双突表现严重的发育缺陷，包括严重萌发延迟。初步结果显示RZ-1B/1C可能整合了ABA及GA的合成代谢来调控萌发。据此，本项目希望以RZ-1B/1C为例，研究转录后调控机制在萌发控制中的作用原理。申请人将明确RZ-1B/1C与已有激素调控网络的关系；通过iCLIP技术定位RZ-1C在萌发过程中的直接RNA底物；明确RZ-1B/1C是否参与调控了转录起始，RNA剪接和RNA降解中的一个或多个过程；初步明确转录后调控机制在萌发过程中是如何被动员利用。

本项目旨在解析两个功能冗余的RNA结合蛋白RZ-1B/1C参与种子萌发调控的分子机制。通过萌发表型分析结合激素及激素抑制剂处理，发现rz-1b/1c的萌发延迟与ABA的合成及信号途径被激活相关。进一步，通过ABA含量测定结合RZ-1B/1C在萌发过程中的表达模式分析，证明了ABA含量的上升是突变体萌发延迟的原因。通过转录组分析结合遗传学杂交实验以及RNA免疫沉淀实验，发现RZ-1B/1C通过直接结合ABA合成关键基因NCED6的新生RNA及染色质，在转录水平及染色质水平抑制其表达，从而完成对萌发的促进作用。最后通过表观组学实验结合免疫共沉淀，发现RZ-1B/1C可能通过结合组蛋白去乙酰化酶复合物，在抑制转录的同时为NCED6基因上H3K27me3甲基化修饰的建立提供前提条件。这些结果在种子萌发的控制机制、ABA合成的控制机制、抑制型组蛋白修饰的建立机制等几个方面具有明确且比较重要的科学意义。和该课题相关的成果一篇发表在Molecular Plant，一篇综述文章发表在Plant Physiology上。此外，大部分通过课题执行得到的实验结果已经总结整理成文，目前正在The Plant Cell期刊二审当中。