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 freeMCP-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.
Original language | English (US) |
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Pages (from-to) | 101-109 |
Number of pages | 9 |
Journal | RNA |
Volume | 26 |
Issue number | 1 |
DOIs | |
State | Published - 2020 |
Externally published | Yes |
Bibliographical note
Funding Information:We thank Dr. S. Cabantous for providing the tripartite split GFP plasmids and Dr. R.H. Singer for providing the phage-ubc-nls-ha-VenusN-IRES-nls-ha-pcp-VenusC and phage-CMV-CFP-12 × MBS–PBS plasmids. This work was supported by the Creative-Pioneering Researchers Program through Seoul National University, the Howard Hughes Medical Institute (HHMI)–Wellcome International Scholar Awards from the Wellcome Trust (208468/Z/ 17/Z), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) (2019R1H1A2039 684). S.Y.P. was supported by IBS-R008-D1 from the Institute for Basic Science.
Publisher Copyright:
© 2020 Park et al.
Keywords
- Fluorescence complementation
- Live-cell imaging
- MS2 system
- Single-molecule imaging