Live-cell imaging of single mRNA dynamics using split superfolder green fluorescent proteins with minimal background.

Sung Young Park,Hyungseok C Moon,Hye Yoon Park

doi:10.1261/rna.067835.118

Sung Young Park, Hyungseok C Moon + Show 1 more

Open Access

https://doi.org/10.1261/rna.067835.118

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Journal: RNA (New York, N.Y.)	Publication Date: Oct 22, 2019
Citations: 22	License type: CC BY 4.0

Affiliation: Seoul National University

Abstract

The MS2 system, with an MS2 binding site (MBS) and an MS2 coat protein fused to a fluorescent protein (MCP–FP), has been widely used to fluorescently label mRNA in live cells. However, one of its limitations is the constant background fluorescence signal generated from free MCP–FPs. To overcome this obstacle, we used a superfolder GFP (sfGFP) split into two or three nonfluorescent fragments that reassemble and emit fluorescence only when bound to the target mRNA. Using the high-affinity interactions of bacteriophage coat proteins with their corresponding RNA binding motifs, we showed that the nonfluorescent sfGFP fragments were successfully brought close to each other to reconstitute a complete sfGFP. Furthermore, real-time mRNA dynamics inside the nucleus as well as the cytoplasm were observed by using the split sfGFPs with the MS2–PP7 hybrid system. Our results demonstrate that the split sfGFP systems are useful tools for background-free imaging of mRNA with high spatiotemporal resolution.

Highlights

Fluorescent proteins (FPs) fused to RNA binding proteins (RBPs) have been widely used for visualizing the transport and dynamics of RNA in living cells (Tyagi 2009; Rath and Rentmeister 2015)
We report that the tripartite superfolder GFP (sfGFP) is suitable for observing mRNA dynamics in the cytoplasm and in the nucleus at a single-molecule resolution
Our results show that the sfGFPbased fluorescence complementation (FC) method is a powerful tool for genetically encoded RNA imaging, providing opportunities for investigators to observe diverse RNA dynamics with high spatiotemporal resolution

Summary

Introduction

Fluorescent proteins (FPs) fused to RNA binding proteins (RBPs) have been widely used for visualizing the transport and dynamics of RNA in living cells (Tyagi 2009; Rath and Rentmeister 2015). We report a real-time background-free imaging method using split superfolder GFPs (sfGFPs) for a nextgeneration RNA probe in living cells.

Results

Conclusion