Regulation of alternative splicing in Arabidopsis thaliana - a combined RNA-Seq and RIP-Seq approach

RNA结合蛋白的转录后调控是植物基因表达的重要控制层。特别是，选择性剪接通过选择性地去除内含子从单个基因产生多个转录异构体。与动物相比，植物中识别内含子的方式似乎有很大的不同。因此，了解高等植物中的调味调控具有重要的意义。该项目的总体目标是全面鉴定由RNA结合蛋白AtGRP7(拟南芥富含甘氨酸的RNA结合蛋白7)和AtGRP8控制的转录后网络。这些蛋白质是哺乳动物异质核糖核蛋白的简化版本。它们受昼夜节律系统的调节，参与植物对病原细菌的防御。我们将通过高通量测序(RNA-Seq)对AtGRP7水平升高或降低的植物中发生变化的选择性剪接事件进行全基因组分析。同时，我们将使用RNA免疫沉淀协议在体内鉴定RNA结合底物，以恢复我们在实验室中建立的GFP标记的AtGRP7和相关RNA(RIP-Seq)。特别是，我们关注这些蛋白质在生物钟输出控制中的作用。我们将通过结合现有算法和在需要时开发新的算法来分析得到的RNA-Seq和RIP-Seq数据。对于这次综合数据分析，我们将建立一个模块分析管道，并将建立一个计算预测和实验验证的迭代周期，从而在下一个周期改进分析，并产生越来越多的实验验证结果。此外，我们将寻找AtGRP7和AtGRP8与其他剪接调控因子的共同靶标，以确定拟南芥中替代的剪接网络，并为揭开高等植物中的剪接密码做出贡献。

Posttranscriptional regulation by RNA-binding proteins emerges as an important layer of control in plant gene expression. In particular, alternative splicing generates multiple transcript isoforms from a single gene by selectively removing introns. There appear to be important differences how introns are recognized in plants compared to animals. Thus, understanding spicing regulation in higher plants is of major interest.The overarching goal of this project is a comprehensive identification of the posttranscriptional networks controlled by the RNA-binding proteins AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) and AtGRP8. These proteins are simplified versions of mammalian heterogeneous nuclear ribonucleoproteins. They are regulated by the circadian timing system and are involved in plant defense against pathogenic bacteria.The proposed project combines two complementary experimental strategies. We will perform genome-wide profiling of alternative splicing events that are altered in plants with elevated or reduced level of AtGRP7 by high throughput sequencing (RNA-Seq). In parallel, we will identify in vivo RNA binding substrates using an RNA immunoprecipitation protocol to recover GFP-tagged AtGRP7 and associated RNAs that we have established in the lab (RIP-Seq). In particular, we focus on the role of these proteins in output control of the circadian clock. We will analyze the resulting RNA-Seq and RIP-Seq data by combining existing algorithms and by developing new ones whenever required. For this integrative data analysis, we will establish a modular analysis pipeline, and we will establish an iterative cycle of computational predictions and experimental validations, leading to an improved analysis in the next cycle and to a growing set of experimentally validated results. We expect this pipeline to be of use to the Arabidopsis community and beyond.Furthermore, we will look for common targets of AtGRP7 and AtGRP8 with other splicing regulators to identify an alternative splicing network in Arabidopsis thaliana and contribute to unravel the splice code in higher plants.