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- W3208889886 abstract "Abstract Background: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The MS2-based RNA imaging system exploits the coat protein (MCP) of MS2 bacteriophage with RNA-binding activity and the RNA hairpin structure MS2 binding sequences (MBS) recognized by MCP to indirectly label mRNAs. Co-expression of fluorescent protein (FP)-fused MCP and target mRNA conjugated with multiple copies of MBS allows for visualizing of mRNA localization by confocal microscopy. To minimize the background fluorescence in cytosol, the nucleus localization sequence was used to sequester the MCP-FP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the application of RNA visualization in the nucleus and sometimes may interfere in detecting fluorescence signals in cytosol, especially in cells with low signal-to-noise ratio. Results: By using the split FP-based approach, we eliminated the background fluorescence derived from MCP-FP not bound to mRNAs. MCP and PCP, a coat protein of PP7 bacteriophages with RNA-binding activity, were fused with the N- or C-terminus of split FPs (FP N or FP C ). On co-expression of MCP-FP N and PCP-FP C with the target mRNA conjugated with multiple copies of the hairpin structure recognized by MCP and PCP, the interaction of RNA and proteins can bring split FPs together to reconstitute functional FPs that allow for visualizing target mRNAs in living cells. We optimized the combinations of MCP- and PCP-FPs with minimal background fluorescence and applied 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." @default.
- W3208889886 created "2021-11-08" @default.
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- W3208889886 date "2021-10-28" @default.
- W3208889886 modified "2023-10-17" @default.
- W3208889886 title "Development of a Split Fluorescent Protein-Based RNA Live-Cell Imaging System to Visualize mRNA Distribution in Plants" @default.
- W3208889886 doi "https://doi.org/10.21203/rs.3.rs-994002/v1" @default.
- W3208889886 hasPublicationYear "2021" @default.
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