Moschou, Panagiotis Nikolaou
- Department of Molecular Sciences, Swedish University of Agricultural Sciences
- Foundation for Research and Technology - Hellas (FORTH)
- University of Crete
Abstract
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 TurboRIP (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 degrees 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.
Keywords
TurboID; Proximity biotinylation; RNA immunopurification; Biomolecular condensates; Liquid-liquid phase separation; Plant stress responses
Published in
Bio-protocol
2026, volume: 16, number: 3, article number: e5587
Publisher: BIO-PROTOCOL
SLU Authors
UKÄ Subject classification
Molecular Biology
Botany
Publication identifier
- DOI: https://doi.org/10.21769/BioProtoc.5587
Permanent link to this page (URI)
https://res.slu.se/id/publ/146411