Spatiotemporal analysis of in vivo RNA-protein interaction

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From synthesis to decay, RNAs dynamically interact with RNA-binding proteins (RBPs) in the cell to orchestrate the transcriptome at the level of pre-mRNA splicing, transport, translation, and RNA decay. The lack of a genome-wide view on RBP in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RBPs. Establishment of individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) in plants in our laboratory has enabled us to obtain a global picture of the in vivo binding landscape of the RBP AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7). Here, plants are irradiated with UV light to stabilize RNA-protein complexes and preserve the in vivo situation. The complexes are immunoprecipitated and libraries are generated from the co-precipitated RNAs for high-throughput sequencing. As reverse transcriptase stalls at the peptide remaining at the crosslink site, binding sites are determined with nucleotide resolution using bioinformatics pipelines. AtGRP7 binds to untranslated regions, coding sequences, and introns, indicating that it regulates its targets at multiple steps of RNA maturation. Furthermore, AtGRP7 shuttles between the nucleus and cytoplasm and thus may exert different regulatory functions in the two subcellular compartments. To further dissect the functions of AtGRP7 in the subcellular compartments, we will determine its target transcripts in the cytoplasm and the nucleus separately. We have shown that AtGRP7 binds to transcripts known to be regulated under cold stress and that AtGRP7 affects alternative splicing in response to low temperature. Therefore, we will perform the iCLIP experiments on the nuclear and the cytoplasmic fractions for plants that are exposed to 4 °C and for control plants kept at 20 °C. Analysis of these data will reveal which transcripts interact preferentially in either compartment. Transcripts bound in both compartments may exhibit a shift in the location of the binding site (untranslated regions, coding sequence, and introns) between the compartments, hinting at different regulatory outcomes of the binding events. Furthermore, we will identify changes of AtGRP7 of binding in response to low temperature. Subsequently, we will unravel how AtGRP7 impacts its targets by testing for an influence on RNA stability, alternative splicing or alternative polyadenylation of the binding targets in the cytoplasm and the nucleus. Furthermore, we will assess the impact of AtGRP7 on physiological responses of the plant to cold exposure by assessing cold adaptation in plants with altered AtGRP7 expression. In parallel, we will work towards improving the bioinformatics to determine binding sites from iCLIP data generated in Arabidopsis and to detect quantitative changes. Overall, this project will pave the way to investigate the dynamics of posttranscriptional networks and their relevance for the low temperature response of Arabidopsis.