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A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat–containing RNA

Abstract

Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat–containing 'toxic RNA' associated with Fragile X–associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.

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Figure 1: RNAs containing CGG-Spinach2 can be imaged in living cells.
Figure 2: Spinach2 is brighter than Spinach owing to improved folding.
Figure 3: Properties of Spinach and Spinach2 in vitro and in bacteria.
Figure 4: 5S-Spinach2 is brighter than 5S-Spinach in mammalian cells.
Figure 5: Imaging RNA foci in COS-7 cells.
Figure 6: The effects of tautomycin and 1a on CGG RNA foci.

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Acknowledgements

We thank K.Y. Wu for her role in developing early improvements in Spinach, G.S. Filonov, W. Song, N. Svensen and J. Paige for useful comments and suggestions, and F. Dardel (Université Paris Descartes) for providing plasmids containing the tRNA scaffold sequence. This work was supported by US National Institutes of Health NINDS NS010249 (S.R.J.), NIGMS F32 GM106683 (R.L.S.), and NIGMS GM079235 (M.D.D.).

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Authors

Contributions

R.L.S., M.D.D. and S.R.J. conceived and designed the experiments, M.D.D. provided compounds and assisted in experiments using 1a and CGG foci, R.L.S. performed experiments and analyzed data, and R.L.S. and S.R.J. wrote the manuscript.

Corresponding author

Correspondence to Samie R Jaffrey.

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Competing interests

S.R.J. and R.L.S. are authors of a patent application (provisional patent USPTO# 61/874,819) related to technology described in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Note (PDF 1314 kb)

Formation of foci after transient transfection.

COS-7 cells were transfected with a plasmid that expresses (CGG)60-Spinach2. At 2 h post-transfection, cells were changed into imaging medium containing 20 μM DFHBI. Images were acquired every 20 min for 6 h. The cell in the lower left side of the image displays initial nucleoplasmic signal, followed by formation of smaller foci that grow larger and brighter throughout the experiment. (MOV 779 kb)

Disaggregation of foci after treatment with tautomycin.

A COS-7 cell expressing (CGG)60-Spinach2 aggregates was treated with 5 μM tautomycin and imaged every 5 min for 2 h. Foci disaggregate, leading to nucleoplasmic (CGG)60-Spinach2 after 1 h. (MOV 232 kb)

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Strack, R., Disney, M. & Jaffrey, S. A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat–containing RNA. Nat Methods 10, 1219–1224 (2013). https://doi.org/10.1038/nmeth.2701

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