Impact of in vivo RNA-protein interaction on the fate of chromatin-associated RNA

• Applicant: Professorin Dr. Dorothee Staiger
• Subject Area: Plant Cell and Developmental Biology ; Plant Biochemistry and Biophysics ; Plant Genetics and Genomics
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 422321065

From synthesis to decay, RNAs interact with RNA-binding proteins (RBPs) to orchestrate RNA processing and function. Arabidopsis thaliana glycine-rich RNA-binding protein 7 (At-GRP7) is involved in circadian timekeeping, temperature response, pathogen defense, and floral transition. At the molecular level, it regulates alternative splicing and processing of miR-NA precursors and long noncoding RNAs. We have determined RNAs bound by AtGRP7 and the corresponding binding sites via Individual-nucleotide resolution crosslinking immunoprecip-itation (iCLIP). Cross-referencing these targets with transcripts mis-regulated in plants consti-tutively overexpressing AtGRP7 or in atgrp7 mutants identified transcripts that are regulated at the level of alternative splicing or RNA stability by direct binding of AtGRP7. As decisions on RNA processing including alternative splicing have to be made on nascent RNAs we hypothe-size that analyzing AtGRP7 binding to chromatin-associated RNAs will provide more detailed insights into the regulatory role of AtGRP7 in RNA processing. For this, we will subject plants expressing the AtGRP7-GFP fusion protein in the atgrp7 mutant background to subcellular fractionation and perform iCLIP on the chromatin-associated fraction, using our sensitive plant iCLIP2 protocol to determine bound RNAs and binding sites. To independently validate the iCLIP targets, we will use enzymatic tagging of AtGRP7 with the adenosine deaminase ADAR, leading to A to I editing in the vicinity of AtGRP7 binding sites which can be read as A to G mutations in RNA-seq (HyperTRIBE). To avoid toxic effects of the hyperactive ADAR variant we will establish an inducible system. For an orthogonal transcriptomic analysis, in parallel we will subject AtGRP7-ox, atgrp7 mutant and wild type plants to cell fractionation and sequence the chromatin-associated transcriptome to identify pre-mRNAs, pri-miRNAs or lncRNAs that are aberrantly processed in plants with altered AtGRP7 levels. Intersecting the binding and the transcriptomic data will provide novel insights into a direct effect of AtGRP7 binding to its targets on their co-transcriptional processing and its regulatory networks. Candi-date targets which undergo alternative splicing in response to binding by AtGRP7 will be vali-dated using splicing reporter minigenes and mutational analysis of the binding sites. Currently, a limitation in defining RBP regulatory networks is the use of mixed tissue that impairs corre-lating binding data with gene expression states. While single-cell sequencing is accessible in plants, no method for tissue- or cell-type specific determination of RBP targets exists. We will develop an inducible cell type specific HyperTRIBE system, using AtGRP7 as paradigm. Overall, we expect that the techniques we will adapt here and their application to AtGRP7 will generate a blueprint generally applicable to plant RBPs and the identification of their regulatory networks.

• DFG Programme: Research Grants