A live-cell assay for the detection of pre-microRNA–protein interactions
| Autor: | Sydney L. Rosenblum, Daniel A. Lorenz, Amanda L. Garner |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0303 health sciences
Cell RNA Computational biology Biology Proteomics Biochemistry Genetics and Molecular Biology (miscellaneous) Biochemistry Protein–protein interaction 03 medical and health sciences 0302 clinical medicine medicine.anatomical_structure Chemistry (miscellaneous) Protein-fragment complementation assay Cytoplasm medicine Bioorthogonal chemistry Molecular Biology 030217 neurology & neurosurgery Function (biology) 030304 developmental biology |
| Zdroj: | RSC Chemical Biology. 2:241-247 |
| ISSN: | 2633-0679 |
| DOI: | 10.1039/d0cb00055h |
| Popis: | Recent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined that this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs. |
| Databáze: | OpenAIRE |
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