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调控的重要环节。特别地，选择性剪接通过选择性去除内含子从单个基因产生多种转录物同种型。与动物相比，植物中内含子的识别方式似乎存在重要差异。因此，了解高等植物中的香料调控是主要interest.The总体目标，本项目是一个全面的识别的RNA结合蛋白AtGRP 7（拟南芥富含甘氨酸的RNA结合蛋白7）和AtGRP 8控制的转录后网络。这些蛋白质是哺乳动物异质核核糖核蛋白的简化版本。它们受昼夜节律计时系统的调节，并参与植物对病原菌的防御。我们将通过高通量测序（RNA-Seq）对AtGRP 7水平升高或降低的植物中发生改变的可变剪接事件进行全基因组分析。与此同时，我们将使用RNA免疫沉淀方案来鉴定体内RNA结合底物，以回收我们在实验室中建立的GFP标记的AtGRP 7和相关RNA（RIP-Seq）。特别是，我们专注于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并在需要时开发新算法来分析所得的RNA-Seq和RIP-Seq数据。对于这种综合数据分析，我们将建立一个模块化的分析管道，我们将建立一个计算预测和实验验证的迭代周期，从而在下一个周期中改进分析，并获得越来越多的实验验证结果。此外，我们还将寻找AtGRP 7和AtGRP 8与其他剪接调节因子的共同靶点，以确定拟南芥中的选择性剪接网络，并为解开高等植物中的剪接密码子做出贡献。

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.