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

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 splicing 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.