Related Experiment Video
Updated: Oct 4, 2025
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Published on: August 6, 2014
Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in
Nien-Chen Huang1, Kai-Ren Luo1, Tien-Shin Yu2
1Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan.
Background:
RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent protein-fused RBP and target mRNA conjugated with corresponding RNA recognition sequences allows for visualizing mRNAs by confocal microscopy. To minimize the background fluorescence in the cytosol, the nuclear localization sequence has been used to sequester the RBP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the visualization of nucleus-localized RNA and sometimes may interfere in detecting fluorescence signals in the cytosol, especially in cells with low signal-to-noise ratio.
Results:
We eliminated the background fluorescence in the nucleus by using the split fluorescent protein-based approach. We fused two different RBPs with the N- or C-terminus of split fluorescent proteins (FPs). Co-expression of RBPs with the target mRNA conjugated with the corresponding RNA recognition sequences can bring split FPs together to reconstitute functional FPs for visualizing target mRNAs. We optimized the system with minimal background fluorescence and used the imaging system to visualize mRNAs in living plant cells.
Conclusions:
We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells.
