Organelle-specific RNA Tagging Probes for Spatially Resolved Profiling of RNA Binding Proteome and Investigation upon Ferroptosis Induction

ABSTRACT: 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 represented an increase of 54.4% and 85.7% compared with previous reports. The probes were further applied in the first omics-level investigation of subcellular-specific RBP-RNA interactions upon ferroptosis induction. Interestingly, large-scale RBPs displayed enhanced interaction with RNAs in nucleus but reduced association with RNAs in cytoplasm during ferroptosis process. Among these RBPs with regulated RNA-binding, translation was found as one of the commonly enriched GO functions by different ferroptosis inducers, indicating ferroptosis could disturb protein translation via different pathways. Furthermore, we discovered dozens of nucleoplasmic translocation candidate RBPs upon ferroptosis induction and validated representative ones by immunofluorescence imaging. The enrichment of TCA cycle in the translocation candidate RBPs may provide new insights for investigating their possible roles in ferroptosis induced metabolism dysregulation. The above findings suggest the potential of our enrichment method for high-throughput RBP mapping with organelle-level spatial resolution.

ORGANISM(S): Homo sapiens

PROVIDER: GSE205553 | GEO | 2022/12/01

REPOSITORIES: GEO