Abstract
BackgroundRNA 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.ResultsWe 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.ConclusionsWe established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells.
Highlights
RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells
We selected two coat proteins (CPs) of MS2 and PP7 bacteriophages, MS2 bacteriophage coat protein (MCP) and PP7 bacteriophage coat protein (PCP), which bind to an RNA hairpin structure of MCP binding sequences (MBS) and PCP binding sequences (PBS), respectively [15]
GFP fluorescence was under the detection limit in cells expressing MCP‐FPN and PCP‐FPC To identify the combinations of MCP and PCP with minimal background GFP fluorescence in plant cells, Fig. 5 GFP background fluorescence was not detected in cells with co-expression of M-FPN and P-FPC
Summary
RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. Direct labeling of mRNAs can be achieved by incorporating in vitro-synthesized mRNA with fluorescein-labeled nucleotides or introducing molecular beacons into cells to hybridize with the target mRNA [6, 7] These in vitro RNA labeling systems require invasive injection of labeled RNA into plant cells such as microinjection or particle bombardment. When NLS-containing MNLS-FPN was coexpressed with P-FPC or FPC-P, weak GFP fluorescence was detected in both the cytosol and nucleus (Fig. 3A– D), which suggests that the separation of MCP and PCP into different subcellular compartments may not significantly reduce the background GFP fluorescence. When P NLS-FPC was co-expressed with M-FPN or NLS-containing MNLS-FPN, the background GFP fluorescence that formed a speckle-like spot was detected only in the nucleus (Fig. 4B, C, E, F, H, I), which suggests that the topology of fusion proteins rather than subcellular localization may contribute to the background GFP fluorescence. The combination of PNLS-FPC with M-FPN or MNLS-FPN produced specklelike background GFP fluorescence in the nucleus, which
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