Defining the RNA interactome by total RNA-associated protein purification.
Mol Syst Biol
; 15(4): e8689, 2019 04 08.
Article
en En
| MEDLINE
| ID: mdl-30962360
ABSTRACT
The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Ribonucleoproteínas
/
Tiouracilo
/
ARN
/
Proteínas de Unión al ARN
Tipo de estudio:
Risk_factors_studies
Idioma:
En
Revista:
Mol Syst Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Reino Unido