Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces.
Mol Cell
; 80(5): 903-914.e8, 2020 12 03.
Article
em En
| MEDLINE
| ID: mdl-33242392
ABSTRACT
Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.
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Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
RNA
/
Proteínas de Ligação a RNA
/
Transcriptoma
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Conformação de Ácido Nucleico
Limite:
Humans
Idioma:
En
Revista:
Mol Cell
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos