GSE203153
Organelle-specific RNA Tagging Probes for Spatially Resolved Profiling of RNA Binding Proteome and Investigation upon Protein Phosphorylation Inhibition
The key role of RNA-binding proteins (RBPs) in posttranscriptional regulation of gene expression is intimately tied to their subcellular localization. Dysregulated localization may severely disrupt the biological functions of RBPs. To reveal the regulatory mechanisms of RBPs, methods for systematically mapping the subcellular localized RBPs and monitoring their translocations under physiological conditions are in high demand. Herein, a subcellular-specific RNA labeling method was developed for efficient enrichment and deep profiling of nuclear and cytoplasmic RBPs. A total of 1221 nuclear RBPs and 1333 cytoplasmic RBPs were enriched and identified using nuclear/cytoplasm targeting enrichment probes, which were over 50% more than that reported in previous works. The probes were further applied in the first large-scale dynamic investigation of subcellular-specific RBPs upon protein phosphorylation inhibition. Six RBPs were found to display cytoplasm-to-nuclear translocation after a treatment with broad-spectrum kinase inhibitors, indicating that phosphorylation is a key factor regulating the organelle orientation of RBPs. The above finding suggests the potential of our enrichment method for high-throughput RBP mapping with organelle-level spatial resolution. Overall design: Comparative gene expression analysis of RNA-seq data from density gradient centrifugation enriched nuclear RNA, BLTF enriched nuclear RNA, density gradient centrifugation enriched cytoplasmic RNA and BETF enriched cytoplasmic RNA in HeLa cells.