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