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Capture of the newly transcribed RNA interactome using click chemistry.
- Source :
-
Nature protocols [Nat Protoc] 2021 Nov; Vol. 16 (11), pp. 5193-5219. Date of Electronic Publication: 2021 Oct 25. - Publication Year :
- 2021
-
Abstract
- Application of synthetic nucleoside analogues to capture newly transcribed RNAs has unveiled key features of RNA metabolism. Whether this approach could be adapted to isolate the RNA-bound proteome (RNA interactome) was, however, unexplored. We have developed a new method (capture of the newly transcribed RNA interactome using click chemistry, or RICK) for the systematic identification of RNA-binding proteins based on the incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV cross-linking and click chemistry-mediated biotinylation. The RNA-protein adducts are then isolated by affinity capture using streptavidin-coated beads. Through high-throughput RNA sequencing and mass spectrometry, the RNAs and proteins can be elucidated globally. A typical RICK experimental procedure takes only 1 d, excluding the steps of cell preparation, 5-ethynyluridine labeling, validation (silver staining, western blotting, quantitative reverse-transcription PCR (qRT-PCR) or RNA sequencing (RNA-seq)) and proteomics. Major advantages of RICK are the capture of RNA-binding proteins interacting with any type of RNA and, particularly, the ability to discern between newly transcribed and steady-state RNAs through controlled labeling. Thanks to its versatility, RICK will facilitate the characterization of the total and newly transcribed RNA interactome in different cell types and conditions.<br /> (© 2021. Springer Nature Limited.)
Details
- Language :
- English
- ISSN :
- 1750-2799
- Volume :
- 16
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Nature protocols
- Publication Type :
- Academic Journal
- Accession number :
- 34697467
- Full Text :
- https://doi.org/10.1038/s41596-021-00609-y