The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells.
J Proteome Res
; 19(7): 2529-2538, 2020 07 02.
Article
em En
| MEDLINE
| ID: mdl-32216351
ABSTRACT
RNA-protein interactions are essential for the regulation of mRNA and noncoding RNA functions and are implicated in many diseases, such as cancer and neurodegenerative disorders. A method that can detect RNA-protein interactions in living mammalian cells on a proteome-wide scale will be an important asset to identify and study these interactions. Here we show that a combination of the mammalian two-hybrid protein-protein detection method KISS (kinase substrate sensor) and the yeast RNA three-hybrid method, utilizing the specific interaction between the MS2 RNA and MS2 coat protein, is capable of detecting RNA-protein interactions in living mammalian cells. For conceptional proof we used the subgenomic flavivirus RNA (sfRNA) of the dengue virus (DENV), a highly structured noncoding RNA derived from the DENV genome known to target host cell proteins involved in innate immunity and antiviral defense, as bait. Using RNA-KISS, we could confirm the previously established interaction between the RNA-binding domain of DDX6 and the DENV sfRNA. Finally, we performed a human proteome-wide screen for DENV sfRNA-binding host factors, identifying several known flavivirus host factors such as DDX6 and PACT, further validating the RNA-KISS method as a robust and high-throughput cell-based RNA-protein interaction screening tool.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA Viral
/
Flavivirus
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Proteome Res
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Bélgica