Visualization and characterization of RNA–protein interactions in living cells
| Autor: | Maria Arroyo, Wen Deng, M. Cristina Cardoso, Ningjun Duan, Heinrich Leonhardt |
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| Rok vydání: | 2021 |
| Předmět: |
AcademicSubjects/SCI00010
RNA-binding protein Biology Green fluorescent protein Narese/16 Mice 03 medical and health sciences Narese/2 0302 clinical medicine Cricetinae Genetics Animals Humans RNA Messenger Epigenetics 030304 developmental biology 0303 health sciences Messenger RNA Rna protein Stem Cells RNA-Binding Proteins RNA In vitro Long non-coding RNA Cell biology Methods Online RNA Long Noncoding 030217 neurology & neurosurgery HeLa Cells Protein Binding |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | RNA–protein interactions are the structural and functional basis of significant numbers of RNA molecules. RNA–protein interaction assays though, still mainly depend on biochemical tests in vitro. Here, we establish a convenient and reliable RNA fluorescent three-hybrid (rF3H) method to detect/interrogate the interactions between RNAs and proteins in cells. A GFP tagged highly specific RNA trap is constructed to anchor the RNA of interest to an artificial or natural subcellular structure, and RNA–protein interactions can be detected and visualized by the enrichment of RNA binding proteins (RBPs) at these structures. Different RNA trapping systems are developed and detection of RNA–protein complexes at multiple subcellular structures are assayed. With this new toolset, interactions between proteins and mRNA or noncoding RNAs are characterized, including the interaction between a long noncoding RNA and an epigenetic modulator. Our approach provides a flexible and reliable method for the characterization of RNA–protein interactions in living cells. Graphical Abstract Graphical AbstractThe basic principle, applications and extensions of RNA trap based RNA-protein interaction assay. |
| Databáze: | OpenAIRE |
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