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Live imaging of mRNA using RNA-stabilized fluorogenic proteins

Nature Methods volume 16, pages 862–865 (2019)Cite this article

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Abstract

Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA–protein complexes that enable live imaging of mRNA in living cells.

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Fig. 1: Design and optimization of an RNA-regulated protein destabilization domain.
Fig. 2: Peptide tDeg confers Pepper RNA-dependent regulation on diverse proteins.
Fig. 3: Imaging green Pepper-tagged β-actin mRNA in living cells.

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Data availability

The sequence of mammalian expression plasmids used in this study is available on GenBank: CMV-mCherry-(F30-2xPepper)10 (MN052904), mini-CMV-(mNeonGreen)4-tDeg (MN052905), CMV-CytERM-mCherry-(F30-2xPepper)10 (MN052906), UbC-(mNeonGreen)4–tDeg (MN052907), pAV-U6 + 27-Tornado-F30-Pepper (TAR Variant-2) (MN052908), pAV-U6 + 27-Tornado-F30-TAR Variant-1 (MN052909). These plasmids are available via Addgene according to the terms of the Uniform Biological Material Transfer Agreement. Source data for Figs. 1–5 and Supplementary Figs. 2–5, 8–12 are available with the paper online.

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Acknowledgements

This work was supported by the National Institutes of Health (grant no. R01NS056306 and U01MH109087 to S.R.J.) and a FRAXA Postdoctoral Fellowship (to J.W.). M.E.T. and D.K. were supported by the Oncode Institute which is partly funded by the Dutch Cancer Society. We thank L. Mei and V. Despic for technical support, the Bio-Imaging Resource Center at Rockefeller University for technical support on imaging experiments, and members of the S.R.J. laboratory, especially X. Li, J. Litke and J.D. Moon, for useful comments and suggestions.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA

    Jiahui Wu, Sara Zaccara, Hyaeyeong Kim & Samie R. Jaffrey

  2. Oncode Institute, Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands

    Deepak Khuperkar & Marvin E. Tanenbaum

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  1. Jiahui Wu
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  2. Sara Zaccara
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  3. Deepak Khuperkar
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  4. Hyaeyeong Kim
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  5. Marvin E. Tanenbaum
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Contributions

J.W. and S.R.J. conceived of and designed the experiments. J.W. performed the experiments and analyzed the data. S.Z. performed mRNA half-life measurements and quantitative PCR with reverse transcription, and generated the Halo-G3BP1 U2OS cell line. D.K. imaged membrane-tethered mRNAs. H.K. designed F30 variants. J.W., S.Z., D.K., M.E.T. and S.R.J. wrote the manuscript.

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Correspondence to Samie R. Jaffrey.

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S.R.J. is a founder, advisor to and owns equity in, Lucerna Technologies.

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Peer review information: Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Design of tDeg, an RNA-regulated destabilization domain.

Shown is a structural representation of how TAR binds to the tDeg, and may therefore obstruct recognition of the Arg-Arg-Arg-Gly degradation-inducing signal. RNA is depicted in grey, and peptide sequence is shown in red. A cartoon representation of RNA binding to the tDeg sequence is shown in (a). Here, we designed a bifunctional peptide sequence, called tDeg (colored in red), that functions both as a destabilization domain and as a binding site for the bovine immunodeficiency virus TAR RNA (colored in grey). Knowing that the TAR RNA binds to specific amino acids in the Tat peptide including the two C-terminal arginines, we added an Arg-Gly (highlighted in a black box) to the C-terminus of the Tat peptide to make the full Arg-Arg-Arg-Gly degron. When the TAR RNA binds to this bifunctional domain, it impedes the function of the destabilization domain by sterically blocking recognition of the Arg-Arg-Arg-Gly degron by proteasomal machinery. The structure model (b) of the Tat-TAR complex shows that the first two arginines of the Arg-Arg-Arg-Gly degron would be inaccessible to any Arg-Arg-Arg-Gly-binding protein that mediates its degradation. The additional Arg-Gly residues are modeled into the C-terminus of Tat in a black box in (b). The structure representation in (b) is based on the NMR structure of the bovine immunodeficiency virus Tat-TAR complex (PDB entry: 1BIV)1. 1. Puglisi, J. D., Chen, L., Blanchard, S. & Frankel, A. D. Solution structure of a bovine immunodeficiency virus Tat-TAR peptide-RNA complex. Science 270, 1200–3 (1995).

Supplementary Figure 2 tDeg confers protein instability to EYFP by proteasomal degradation.

In Fig. 1b, we showed that tDeg confers protein instability to EYFP. However, the lack of yellow fluorescence of EYFP-tDeg in Fig. 1b could be due to protein misfolding or aggregation. Here, we examined whether the lack of yellow fluorescence of EYFP-tDeg is due to proteasomal degradation. In these experiments, HEK293T cells were transiently transfected with a plasmid expressing EYFP-tDeg. These cells were then treated with vehicle (DMSO) or a proteasome inhibitor (10 μM MG132) for 7 hours, respectively. When treated with vehicle (DMSO), minimal yellow fluorescence was detected. This result is consistent with the result from Fig. 1b. However, when proteasome activity was inhibited by treatment of 10 μM MG132 for 7 hours, the yellow fluorescence of EYFP-tDeg was restored. Thus, this confirmed that the tDeg tag markedly reduces the stability of EYFP by inducing its proteasomal degradation. All cells were stained with Hoechst dye. Scale bar, 40 μm. In (b), normalized total cellular yellow fluorescence of individual cells is plotted (n = 3 independent cell cultures). Values are means ± s.d. ****P = 5.6 x 10-36 by unpaired two-tailed Student’s t-test.

Supplementary Figure 3 Engineered TAR variants’ higher efficiency in stabilizing EYFP-tDeg proteins were not due to expression differences in EYFP-tDeg mRNA or the circular TAR RNAs.

In Fig. 1b, c, we showed that circular wild-type TAR, Variant-1, and Variant-2 showed 24-fold, 36-fold, and 38-fold fluorescence increases, respectively. However, the improved efficiency in stabilizing EYFP-tDeg protein could be due to uneven expression levels of the EYFP-tDeg mRNA, or the uneven expression levels of the circular TAR RNA variants. Here, we compared the relative expression of EYFP-tDeg mRNA (a) and the relative expression of circular TAR RNA variants (b). In these experiments, HEK293T cells were transiently transfected with a plasmid expressing EYFP-tDeg and the corresponding circular TAR RNA variant as shown in Fig. 1b, c. Totally RNA was extracted by Trizol extraction. EYFP-tDeg mRNA expression level was quantified using RT-qPCR. Each circular TAR RNA variant’s expression level was quantified by running the extracted total RNA on a TBE-Urea gel followed by SYBR™ Gold nucleic acid gel staining. These results showed that there is no significant expression difference in the EYFP-tDeg mRNA or the circular TAR RNA variants. Thus, this confirmed that the engineered circular TAR RNA variants indeed show higher efficiency in stabilizing tDeg-tagged EYFP. Data were collected from 2 independent cell cultures. Values are means ± s.d.

Supplementary Figure 4 tDeg can be regulated by the Pepper RNA aptamer in diverse mammalian cell types.

In Fig. 1, we showed that EYFP-tDeg can be regulated by the Pepper RNA aptamer in HEK293T cells. Here, we examined whether tDeg can be regulated by the Pepper RNA aptamer in various mammalian cell types (a). In these experiments, U2OS cells (b, e), COS-7 cells (c, f), or HeLa cells (d, g) transiently expressed EYFP-tDeg with and without the circular Pepper RNA aptamer, respectively. In each case, cells showed low or undetectable levels of yellow fluorescence without the circular Pepper RNA aptamer. The yellow fluorescence of EYFP-tDeg was only restored when the circular Pepper RNA aptamer was coexpressed. Thus, tDeg can be regulated by the Pepper RNA aptamer in diverse mammalian cell types. All cells were stained with Hoechst dye. Scale bar, 20 μm. Normalized total cellular fluorescence (e, f and g) of individual cells is plotted (n = 3 independent cell cultures). Values are means ± s.d. ****PU2OS = 5.7 x 10-59; ****PCOS-7 = 1.6 x 10-46; ****PHeLa = 2.0 x 10-139 by unpaired two-tailed Student’s t-test.

Supplementary Figure 5 tDeg confers Pepper RNA-dependent regulation to diverse proteins.

In Fig. 2, we showed that tDeg confers Pepper RNA-dependent regulation of different fluorescent proteins and the luciferase, NanoLuc2. Here we tested whether tDeg confers Pepper-dependent regulation to proteins with different functions and localizations in cells (a). In these experiments, HEK293T cells transiently expressed EGFP-TetR-tDeg (b, e), EGFP-EZH2-tDeg (c, f), or mCherry-NF-κB-tDeg (d, g), with and without the circular Pepper RNA aptamer, respectively. In each case, proteins were nearly undetectable unless coexpressed with the circular Pepper RNA. Furthermore, we compared protein localization of these proteins without tDeg and the circular Pepper RNA to their stabilized counterparts by tDeg and circular Pepper RNA. We observed that EGFP-TetR-tDeg with circular Pepper RNA showed more green fluorescent signals in the cytosol compared to EGFP-TetR. We did not observe significant change of protein localization in the case of EGFP-EZH2-tDeg or mCherry-NF-κB-tDeg with the circular Pepper RNA. We concluded that tDeg is a versatile tag for RNA-dependent protein stabilization. All cells were stained with Hoechst dye. Scale bar, 40 μm. Normalized total cellular fluorescence (e, f and g) of individual cells is plotted (n = 3 independent cell cultures). Values are means ± s.d. ****PEGFP-TetR-tDeg = 2.9 x 10-136; ****PEGFP-EZH2-tDeg = 1.1 x 10-120; ****PmCherry-NF-κB-tDeg = 3.5 x 10-119 by unpaired two-tailed Student’s t-test. 2. Hall, M. P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–57 (2012).

Supplementary Figure 6 Optimization of a concatenated Pepper tag to image mRNAs in live cells.

We sought to use Pepper RNA-regulated fluorogenic proteins to fluorescently tag mRNAs in live cells. As a first step, we sought to determine the best way to incorporate the Pepper aptamers in the 3’UTR of a transcript of interest. In these experiments, we expressed a fluorogenic protein (mNeonGreen)2-tDeg and an mCherry mRNA reporter containing 3’UTR tags comprising 10 or 20 concatenated Pepper aptamers with and without a folding scaffold, F30, respectively. In the case of the (Pepper)20 and (F30-2xPepper)10 tags, mobile green fluorescent puncta in the cytosol were observed. A signal to noise ratio was evident when the (F30-2xPepper)10 tag (signal to noise ratio = 1.8) was used, compared to the (Pepper)20 tag (signal to noise ratio = 1.5). However, puncta were not readily detectable with either the (Pepper)10 tag or the (F30-1xPepper)10 tag. Therefore, we used the (F30-2xPepper)10 tag to image mRNAs in the subsequent experiments. Scale bar, 20 μm. This experiment was performed three times with similar results.

Supplementary Figure 7 Design of Pepper tags for imaging mRNA.

Design and sequences of four Pepper tags used in Supplementary Fig. 6: (Pepper)10 (a), (F30-1xPepper)10 (b), (Pepper)20 (c), and (F30-1xPepper)10 (d).

Supplementary Figure 8 Optimization of the number of fluorogenic mNeonGreen monomers in the fluorogenic protein for imaging mRNA in live cells.

In Supplementary Fig. 6, we observed that (F30-2xPepper)10 is the optimal tag for imaging mRNAs in live cells. To further optimize the system of using Pepper RNA-regulated fluorogenic protein to image mRNAs, we sought to increase the fluorescence signal to background noise ratio of the mobile green fluorescent puncta by increasing the number of fluorogenic mNeonGreen. In these experiments, we transiently expressed an mCherry mRNA reporter tagged with (F30-2xPepper)10, and tandem fluorogenic mNeonGreen with 2, 3, or 4 copies, respectively, in cells. Here, we observed an increase of fluorescence intensity of the green fluorescent puncta as the number of tandem mNeonGreen increased from 2, 3, to 4 copies, respectively (b) and (c). We also re-tested mRNAs tagged with (F30-1xPepper)10 using the (mNeonGreen)4-tDeg fluorogenic protein. We showed that puncta were detectable, but not as pronounced as when the (F30-2xPepper)10 tag was used. Thus, we concluded that (mNeonGreen)4-tDeg provides a high signal to noise ratio for imaging mRNAs. Scale bar, 20 μm. (c) Fluorescence intensity of green fluorescent puncta of individual cells is plotted (n = 3 independent cell cultures). Values are means ± s.d. ****P(Pepper)20:(F30-2xPepper)10 = 4.6 x 10-19; ****P(mNeonGreen)2-tDeg:(mNeonGreen)3-tDeg = 7.7 x 10-9; ****P(mNeonGreen)2-tDeg:(mNeonGreen)4-tDeg = 2.5 x 10-29; ****P(mNeonGreen)3-tDeg:(mNeonGreen)4-tDeg = 2.0 x 10-9; ****P(F30-2xPepper)10:(F30-1xPepper)10 = 5.6 x 10-17 by one-way ANOVA.

Supplementary Figure 9 Pepper tag enables visualization of both nuclear and cytosolic mRNAs.

a, DNA plasmid constructs used for imaging mRNAs in the nucleus and cytosol. b, To image nascent transcription of mRNA, we imaged cells coexpressing an mCherry mRNA reporter containing a 3’UTR green Pepper mRNA tag, (F30-2xPepper)10, and a green fluorogenic protein, (mNeonGreen)4-tDeg. We observed cytosolic green fluorescent puncta reflecting mCherry mRNA transcripts and nuclear green fluorescent puncta, potentially reflecting mCherry mRNA transcripts. We noticed there were less green fluorescent puncta in the nucleus compared to the cytosol. This potentially reflects that most of the nuclear mCherry mRNA transcripts were exported out of the nucleus. Scale bar, 20 μm. c, Summary data of cytosolic and nuclear mRNA fluorescence intensity in (b) (n = 201 fluorescent puncta). Values are means ± s.d. This experiment was performed three times with similar results.

Supplementary Figure 10 Pepper tag and fluorogenic protein enable visualization of individual mRNAs.

To examine whether the puncta observed when imaging Pepper-tagged mRNAs might be stable degradation intermediates, we performed northern blot on total RNA extracted from cells expressing (F30-2xPepper)10-tagged mCherry RNA transcripts with and without coexpressing the fluorogenic protein, (mNeonGreen)4-tDeg. In these experiments, only full-length mRNA transcript was detected (a). Therefore, we concluded that the fluorescent puncta in cells largely reflects the full-length transcript, and that degraded or liberated Pepper aptamers do not accumulate in cells. To assess whether the mobile green fluorescent puncta seen in cells expressing Pepper-tagged mRNA represent single mRNAs, we used a previously described mRNA imaging method in which the resulting puncta were validated to represent single mRNAs3. This system uses 24 PP7 RNA hairpins in the 3’UTR of a reporter mRNA, and a 3xmCherry-CAAX protein fused to PCP (PP7 coat protein), the PP7-binding protein. The PCP-3xmCherry-CAAX protein is anchored to the membrane via the CAAX sequence, which reduces puncta motility and facilitates quantitative fluorescence measurements. We imaged a PP7-containing reporter mRNA with and without the (F30-2xPepper)10 tag (b). The (mNeonGreen)4-tDeg fluorogenic protein was used to image the Pepper-tagged mRNAs. If the Pepper tag or the green fluorogenic protein causes mRNA to aggregate, we would expect to observe the Pepper-tagged reporter mRNA puncta to have higher red fluorescence (from PCP-3xmCherry-CAAX) compared to the reporter mRNA puncta without the Pepper tag. The results of these experiments showed that the red fluorescence intensity distribution of the reporter mRNA is not significantly different with and without the Pepper tag (c) (Black bars, 19 cells, 485 mRNAs; Red bars, 13 cells, 384 mRNAs). This suggests that the Pepper tag and the green fluorogenic protein do not cause mRNA aggregation. Furthermore, we observed colocalization between the green and magenta fluorescent puncta only when the reporter mRNA contained the Pepper tag (d). These results suggest that the green fluorescent puncta observed using the Pepper tag and green fluorogenic protein are indeed individual mRNAs. Scale bar, 5 μm (left panel in d), 1 μm (right panel in d). In (d), the experiment of reporter mRNA with Pepper was performed three times with similar results, the experiment of reporter mRNA without Pepper was performed twice with similar results. 3. Yan, X., Hoek, T. A., Vale, R. D. & Tanenbaum, M. E. Dynamics of Translation of Single mRNA Molecules In Vivo. Cell 165, 976–89 (2016).

Supplementary Figure 11 Pepper tag and fluorogenic protein do not have observable effects on mRNA turnover kinetics, mRNA translation efficiency, or proteasome activity in cells.

To test whether adding the Pepper tag to an mRNA transcript affects its stability, we constructed reporter plasmids expressing mCherry transcripts with and without the (F30-2xPepper)10 tag. HEK293T cells were transfected with these two reporter plasmids, respectively. In each case, the same cells were cotransfected with the (mNeonGreen)4-tDeg fluorogenic protein. The cells were treated with 5 μg/mL actinomycin D to inhibit new transcription. The amount of reporter mRNA transcripts remaining at each time point was quantified by RT-qPCR at t = 0, 1, 2, 4, and 6 hours of actinomycin D treatment. The results showed that fusing the Pepper tag to the reporter mRNA (half-life = 5.9 hours) does not significantly affect its turnover rate compared to its untagged counterpart (half-life = 6.0 hours) (a). Thus, these data suggest that Pepper-tagged mRNA transcripts have similar turnover kinetics as mRNAs without the Pepper tag. Data were collected from 2 independent cell cultures. Values are means ± s.d. To test whether adding the Pepper tag to an mRNA transcript affects its protein translation efficiency, we compared the protein translation efficiency of an mCherry mRNA with and without the (F30-2xPepper)10 Pepper tag. HEK293T cells expressing mCherry mRNA or mCherry-(F30-2xPepper)10 mRNA were harvested. We quantified the amount of mCherry protein and mCherry mRNA by western blotting and RT-qPCR, respectively. We observed a slight decrease of mRNA levels in the Pepper-tagged mCherry mRNA compared to its untagged counterpart (c). The same phenomenon was also observed in the mCherry mRNA tagged with the 24xMS2 hairpins4. This may due to the longer transcript length associate with 3’UTR-tagged mRNAs. We calculated protein translation efficiency by normalizing the amount of mCherry protein to the amount of mCherry mRNA (b-d). We found that there is no significant difference in protein translation efficiency between the untagged mCherry mRNA transcript and the Pepper-tagged mCherry mRNA transcript (d). These results suggest that Pepper tag does not significantly affect protein translation of these mRNA reporter transcripts. Data were collected from 2 independent cell cultures. Values are means ± s.d. Since the degradation mechanism of the fluorogenic proteins relies on ubiquitination and subsequent proteasomal degradation, expression of fluorogenic proteins could lead to the overload of proteasome activity in cells. To test whether the expression of fluorogenic proteins overloads proteasome activity, we expressed a fluorogenic protein, (mNeonGreen)4-tDeg in HEK293T cells. If the expression of (mNeonGreen)4-tDeg overloads the activity of the proteasome, we would expect to observe an accumulation of the ubiquitinated protein in cells. Here, we showed western blotting results using an anti-ubiquitin antibody of untransfected cells and cells expressing (mNeonGreen)4-tDeg. We did not observe significant difference in the ubiquitinated proteins (e). As a control, untransfected cells treated with a proteasome inhibitor (10 μM MG132) for 5 hours showed a significant increase of the ubiquitinated proteins (e). Thus, these results suggest that expression of fluorogenic proteins does not overload proteasome activity in cells. Data shown here is a representative image from 2 independent cell cultures. 4. Wu, B. et al. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev. 29, 876–86 (2015).

Supplementary Figure 12 Pepper tag does not disrupt the localization of mRNAs.

To determine whether the Pepper tag disrupts an mRNA’s proper cellular localization, we chose an ER-targeting reporter mRNA, and imaged its localization in cells using the (F30-2xPepper)10 Pepper tag and the (mNeonGreen)4-tDeg fluorogenic protein (a). This ER-targeting reporter mRNA encodes the first 29 amino acids of cytochrome p450, CytERM, and the encoding sequence of mCherry followed by (F30-2xPepper)10 in the 3’UTR (a). During protein translation, the CytERM peptide will direct this reporter mRNA to the outer ER membrane, and confine the mRNA’s mobility. Indeed, we observed green fluorescent puncta with low mobility (b, d), suggesting that the reporter mRNA is localized to the outer ER membrane. To further validate the localization of the ER-targeting reporter mRNA, we treated the cells with a translation inhibitor (100 μg/mL, puromycin) to liberate the reporter mRNA from the ER into the cytosol. We observed a significant mobility increase of the green fluorescent puncta (c, d), reflecting the dissociation of the reporter mRNA from the ER. Together, these results confirmed that the Pepper tag does not disrupt the localization of mRNAs. Scale bar in (b, c), 10 μm. Relative diffusion coefficient of mRNA puncta is plotted (n = 2 independent cell cultures). Values are means ± s.d. ****P = 2.7 x 10-6 by unpaired two-tailed Student’s t-test.

Supplementary Figure 13 (mNeonGreen)4-tDeg without the Pepper-tagged β-actin mRNA does not accumulate in stress granules upon arsenite treatment.

In Fig. 3, we showed that cytosolic green fluorescent puncta accumulated in stress granules to form foci upon application of 500 µM arsenite. However, the formation of green fluorescent foci in stress granules could be due to aggregation of the fluorogenic protein, (mNeonGreen)4-tDeg, regardless of the present of the β-actin mRNA. To test whether this is the case, we coexpressed (mNeonGreen)4-tDeg with circular Pepper RNA in U2OS cells (a). Before arsenite treatment, we observed cytosolic green fluorescent without any puncta, which is consistent with our results in Supplementary Fig. 4. Upon application of 500 µM arsenite, we did not observe any green fluorescent foci formation (b). These results confirmed that the formation of green fluorescent foci in Fig. 3 was indeed due to the β-actin mRNA. This experiment was performed twice with similar results. Scale bar, 20 μm.

Supplementary Figure 14 Imaging mRNAs using Pepper RNA-regulated fluorogenic proteins with different hues.

So far, we described mRNA imaging using the green Pepper RNA tag, comprising the Pepper aptamer and a fluorogenic mNeonGreen protein. To further expand the color palette for mRNA imaging, we expressed (mVenus)2-tDeg and (mCherry)2-tDeg to generate yellow Pepper and red Pepper complexes on mRNA. In these experiments, we used (mVenus)2-tDeg to image an mCherry mRNA reporter tagged with (F30-2xPepper)10 (a), and we used (mCherry)2-tDeg to image a β-actin mRNA reporter tagged with (F30-2xPepper)10 (b), respectively. In both cases, we observed mobile fluorescent puncta in cells. This experiment was performed twice with similar results. Scale bar, 20 μm.

Supplementary Figure 15 Uncropped blots

(a) Northern blot for Supplementary Fig. 10a (b) Western blot for Supplementary Fig. 11e, anti-ubiquitin (c) Western blot for Supplementary Fig. 11e, anti-GAPDH

Supplementary information

Supplementary Information

Supplementary Figs. 1–15, Supplementary Tables 1–2 and Supplementary Protocol.

Reporting Summary

Supplementary Video 1

(F30-2xPepper)10 tag enables visualization of mRNAs in live cells.

Supplementary Video 2

Puromycin treatment liberated the Pepper-tagged reporter mRNA from the ER and increased its mobility.

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Wu, J., Zaccara, S., Khuperkar, D. et al. Live imaging of mRNA using RNA-stabilized fluorogenic proteins. Nat Methods 16, 862–865 (2019). https://doi.org/10.1038/s41592-019-0531-7

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