Capture of the newly transcribed RNA interactome using click chemistry.
Nat Protoc
; 16(11): 5193-5219, 2021 11.
Article
en En
| MEDLINE
| ID: mdl-34697467
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN
/
Química Clic
Límite:
Humans
Idioma:
En
Revista:
Nat Protoc
Año:
2021
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Reino Unido