Turbo-RIP: A Protocol for TurboID-based RNA Immunopurification to Map RNA Landscapes in Plant Biomolecular Condensates.

Biomolecular condensates organize cellular processes through liquid-liquid phase separation, creating membrane-less compartments enriched in specific proteins and RNAs. Understanding their RNA composition is essential for elucidating plant stress responses, yet capturing these transiently associated RNAs remains technically challenging. We present Turbo-RIP (TurboID-based proximity labeling with RNA immunopurification), a comprehensive protocol for identifying condensate-associated RNAs in plants. Turbo-RIP employs the biotin ligase TurboID to label proximal proteins at 22 °C, followed by formaldehyde crosslinking and streptavidin-based capture of protein-RNA complexes. We provide detailed procedures for three cloning strategies, transformation of Nicotiana benthamiana and Arabidopsis thaliana, validation of TurboID activity, and RNA recovery. The protocol successfully captured processing body-associated RNAs with minimal background. Turbo-RIP enables systematic mapping of RNA populations within plant condensates under diverse conditions. The protocol requires 3-5 days from sample preparation to RNA isolation, with construct validation taking 2-4 weeks. All procedures use standard laboratory equipment, making Turbo-RIP accessible for plant molecular biology laboratories. Key features • Apply TurboID-based proximity labeling specifically for RNA capture in plant condensates. • Optimize experimental conditions for different plant species and condensate types. • Implement quality control measures and data analysis pipelines.