Meyer Katja, Lewinski Martin, Köster Tino, Staiger Dorothee
RNA Biology and Molecular Biology, Faculty of Biology, Bielefeld University, Bielefeld, Germany.
Sci Data. 2025 Aug 6;12(1):1374. doi: 10.1038/s41597-025-05716-z.
RNA-binding proteins impact RNA processing including splicing, translation and RNA decay to dictate the fate of RNAs. Thus, insights into RNA-protein interactions in the cell provide insights into regulatory networks regulating gene expression at the posttranscriptional level. In higher plants, only few RNA-binding proteins have their in vivo targets and binding sites determined, mostly due to technical challenges posed by plant tissue. We previously adapted individual nucleotide-resolution crosslinking and immunoprecipitation that relies on UV-light irradiation to stabilize RNA-protein interactions in the cell to Arabidopsis plants. Here, we profiled the in vivo binding landscape of the glycine-rich RNA-binding protein AtGRP8 by iCLIP. Transgenic plants expressing AtGRP8 fused to GREEN FLUORESCENT PROTEIN under the native promoter were subjected to UV-crosslinking. RNA-protein complexes were immunoprecipitated and bound RNAs were determined by RNA-seq. A bioinformatics pipeline tailor-made for Arabidopsis identified target RNAs, and delineated binding sites and binding motifs for AtGRP8. These data will aid in assembling a compendium of binding sites for plant RNA-binding proteins and contribute to unravel plant posttranscriptional networks.
RNA结合蛋白影响包括剪接、翻译和RNA降解在内的RNA加工过程，从而决定RNA的命运。因此，深入了解细胞中的RNA-蛋白质相互作用有助于深入了解在转录后水平调控基因表达的调控网络。在高等植物中，只有少数RNA结合蛋白的体内靶点和结合位点得到确定，这主要是由于植物组织带来的技术挑战。我们之前将依赖紫外线照射以稳定细胞中RNA-蛋白质相互作用的单核苷酸分辨率交联和免疫沉淀技术应用于拟南芥植株。在此，我们通过iCLIP分析了富含甘氨酸的RNA结合蛋白AtGRP8的体内结合图谱。对在天然启动子控制下表达与绿色荧光蛋白融合的AtGRP8的转基因植株进行紫外线交联。免疫沉淀RNA-蛋白质复合物，并通过RNA测序确定结合的RNA。为拟南芥量身定制的生物信息学流程鉴定了靶RNA，并描绘了AtGRP8的结合位点和结合基序。这些数据将有助于汇编植物RNA结合蛋白的结合位点简编，并有助于揭示植物转录后网络。
