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Issue 1, 2021
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A live-cell assay for the detection of pre-microRNA–protein interactions

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Abstract

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.

Graphical abstract: A live-cell assay for the detection of pre-microRNA–protein interactions

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Supplementary files

Article information


Submitted
28 Apr 2020
Accepted
26 Nov 2020
First published
08 Dec 2020

This article is Open Access

RSC Chem. Biol., 2021,2, 241-247
Article type
Paper

A live-cell assay for the detection of pre-microRNA–protein interactions

S. L. Rosenblum, D. A. Lorenz and A. L. Garner, RSC Chem. Biol., 2021, 2, 241
DOI: 10.1039/D0CB00055H

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