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An improved imaging system that corrects MS2-induced RNA destabilization

Nature Methods volume 19, pages 1558–1562 (2022)Cite this article

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Abstract

The MS2 and MS2-coat protein (MS2-MCP) imaging system is widely used to study messenger RNA (mRNA) spatial distribution in living cells. Here, we report that the MS2-MCP system destabilizes some tagged mRNAs by activating the nonsense-mediated mRNA decay pathway. We introduce an improved version, which counteracts this effect by increasing the efficiency of translation termination of the tagged mRNAs. Improved versions were developed for both yeast and mammalian systems.

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Fig. 1: MBS tagging may destabilize mRNAs through the NMD pathway in yeast.
Fig. 2: The improved MS2-MCP system minimizes mRNA destabilization in yeast and mammalian cells.

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Data availability

All of the data and reagents used in this study are available upon request. The key plasmids of the MS2-MCP systems V8 and V9 will be available through Addgene. Source data are provided with this paper.

Code availability

The MATLAB scripts used to measure the distance between mRNAs and mitochondria are available at the GitHub repository (https://github.com/WeihanLi-biology/An-improved-MS2-MCP-imaging-system-with-minimal-perturbation-of-mRNA-stability).

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Acknowledgements

The authors thank A. Jacobson, U.T. Meier, R.A. Coleman and the members of the Singer laboratory for insightful discussions. The authors also thank X. Meng for her help with cloning. This work was supported by American Heart Association Postdoctoral Fellowship 903024 (W.L.), 1R35 GM136296-01 (R.H.S.) and the European Research Council ERCStG-714739 IlluMitoDNA (C.O.).

Author information

Author notes

  1. These authors contributed equally: Weihan Li, Anna Maekiniemi.

Authors and Affiliations

  1. Program in RNA Biology, Albert Einstein College of Medicine, The Bronx, NY, USA

    Weihan Li, Anna Maekiniemi, Hanae Sato & Robert H. Singer

  2. Department of Cell Biology, Albert Einstein College of Medicine, The Bronx, NY, USA

    Weihan Li, Anna Maekiniemi, Hanae Sato & Robert H. Singer

  3. Faculty of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Christof Osman

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Contributions

W.L. and A.M. designed and performed the experiments and analyzed the data. H.S. provided guidance in generating mammalian cell lines expressing MCP-GFP-eRF3/PABPC1*. C.O. provided the analysis pipeline to determine the distance between mitochondria and mRNA. W.L., A.M. and R.H.S. conceived the study and wrote the manuscript with input from H.S. and C.O.

Corresponding authors

Correspondence to Weihan Li, Anna Maekiniemi or Robert H. Singer.

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The authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks Luisa Romão and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 The abundance of yeast MDN1, CLB2, ATP3, and ATP4 mRNAs are not reduced by MBS tagging.

(a) RT–qPCR of WT and MBS-tagged mRNAs. The mRNA levels were normalized to their corresponding WT mRNAs. n = 3 biologically independent experiments. Error bars indicate mean ± SD. ns, not significant (two-sided Student’s t-test). (b) Representative smFISH images of CLB2 mRNA (green). DNA was stained with DAPI (blue). Scale bars are 2 µm. (c) The number of CLB2 mRNAs per cell as quantified from the smFISH images. Each dot corresponds to one individual cell. The number of cells being analyzed are 658 (WT) and 1060 (CLB2-24×MBS). Three replicate experiments were performed. Error bars indicate mean ± SD.

Extended Data Fig. 2 Altering the location of the MBS array does not restore the ATP2 mRNA abundance.

(a) Illustration of the MBS-tagging positions on ATP2 mRNA. (b) RT–qPCR of the ATP2 mRNA in the indicated strains. The mRNA levels were normalized to the WT ATP2 mRNA level. Data were analyzed from three replicate experiments. The P values are <0.0001, 0.0001. Error bars indicate mean ± SD. ***P ≤ 0.001, ****P ≤ 0.0001 (two-sided Student’s t-test).

Extended Data Fig. 3 MBS tagging decreases the mRNA stability of HAC1 and PMA1.

(a–c) mRNA stability assay of HAC1 (A), PMA1 (B), and ACT1 (C). Cells were treated with phenanthroline to inhibit transcription. In the experiments measuring HAC1 mRNA stability, cells were treated with 1 μg/ml tunicamycin for 2 h before adding phenanthroline. mRNA levels at the indicated time points were measured by RT–qPCR. A two-sided Student’s t-test was performed to compare WT and 24×MBS strains at the indicated time points. In (A), the P values from left to right are 0.0066, 0.0031, 0.0048. In (B), the P values from left to right are 0.031, 0.091, 0.59. *P ≤ 0.05, **P ≤ 0.01; ns, not significant. n = 3 biologically independent experiments. Error bars indicate mean ± SD.

Extended Data Fig. 4 The abundance of yeast Atp2 protein is reduced by MBS tagging and restored by expressing MCP-GFP-SUP35 or MCP-GFP-PAB1*.

Western blot analysis of Atp2 proteins in the indicated strains. Atp2 protein was c-terminally tagged with myc epitope. Pgk1 protein was used as a loading control. One representative image is shown from three replicate experiments.

Source data

Extended Data Fig. 5 MCP-GFP-PAB1* can be used to image MBS-tagged ATP2 mRNAs.

(a) Live-cell imaging of yeast cells expressing MCP-GFP-PAB1 (left) or MCP-GFP-PAB1* (right). Images were max-Z projected. Scale bars are 2 µm. The cell outline is marked with a white dashed line. (b, c) Sequence alignment of PAB1 homologs flanking the two conserved phenylalanines, which are F170 and F366 in yeast PAB1 (F142 and F337 in human PABPC1). These two phenylalanines were mutated to valines in PAB1*/PABPC1*.

Extended Data Fig. 6 Expression of MCP-GFP-SUP35 or MCP-GFP-PAB1* restores the stability of HAC1 and PMA1 mRNA.

(a–c) mRNA stability assay of the HAC1 (A), PMA1 (B), and ACT1 (C) mRNA. Experimental conditions were the same as Extended Data Fig. 3. n = 3 biologically independent experiments. A two-sided Student’s t-test was performed to compare 24×MBS and 24×MBS + MCP-GFP-SUP35 strains at the indicated time points. *P ≤ 0.05, **P ≤ 0.01; ns, not significant (Student’s t-test). In (A), the P values from left to right are 0.0074, 0.0020, 0.0039. In (B), the P values from left to right are 0.014, 0.045, 0.15. Error bars indicate mean ± SD. (d) RT–qPCR of ACT1 mRNAs. The mRNA levels were normalized to the WT mRNA level. n = 3 biologically independent experiments. Error bars indicate mean ± SD. ns, not significant (two-sided Student’s t-test).

Extended Data Fig. 7 Tracking single-molecule ATP2 mRNAs using the improved MS2-MCP system.

The original movie is shown in Supplementary Movie 1. Mitochondria were labeled using mitochondria-targeted mKate2 (red). MBS-tagged ATP2 mRNAs were imaged using MCP-GFP-SUP35. The movie was acquired at 20 frames per second in one z plane. The figure shows the molecular trajectories of the mRNAs that were tracked for at least 40 consecutive frames (white arrowed lines). The cell outline is marked with a white dashed line. Scale bar is 2 µm.

Extended Data Fig. 8 smFISH images of P21 mRNAs in U2OS cells.

Representative images of P21 mRNA (green). DNA was stained with DAPI (blue). To induce the expression of P21, U2OS cells were treated with 10 μM Nutlin-3 for 2 h. Scale bars are 4 µm. Two replicate experiments were performed.

Extended Data Fig. 9 Knock-down of UPF1 mRNA in U2OS cells.

RT–qPCR of UPF1 mRNA in the presence or absence of the UPF1 shRNA. n = 3 biologically independent experiments. Error bars indicate mean ± SD. ***P ≤ 0.001 (two-sided Student’s t-test). The P value is 0.0007.

Extended Data Fig. 10 Expression of MCP-GFP-eRF3 or MCP-GFP-PABPC1* restores the mRNA and protein abundance of P21 in U2OS cells.

(a) Increasing levels of MCP-GFP-PABPC1* better restored the P21 mRNA abundance. Cells expressing different levels of MCP-GFP-eRF3 or MCP-GFP-PABPC1* were sorted by FACS (L: low; M: medium; H: high). The level of the P21 mRNA was examined by RT–qPCR. n = 3 biologically independent experiments. Error bars indicate mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; ns, not significant (two-sided Student’s t-test). The P values from left to right are 0.0037, 0.0006, 0.010, <0.0001, 0.0080, 0.0018, 0.015. (b) Western blot of p21 protein. To induce the expression of P21, U2OS cells were treated with 10 μM Nutlin-3 for 24 h. A monoclonal antibody against p21 was used. β-Actin was used as a loading control. One representative image is shown from three replicate experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–5 and legends of Supplementary Movies 1–5.

Reporting Summary

Supplementary Video

Supplementary Movie 1. Live-cell imaging of ATP2 mRNAs using MCP-GFP-SUP35. Mitochondria (red) were labeled using a mitochondria-targeted mKate2. MBS-tagged ATP2 mRNAs (green) were imaged with MCP-GFP-SUP35. The movie was acquired at 20 frames s−1 in one z plane. Scale bar. 2 µm.

Supplementary Video

Supplementary Movie 2. Live-cell imaging of ATP2 mRNAs using MCP-GFP-SUP35. The experimental condition is the same as in Supplementary Movie 1. Scale bar, 2 µm.

Supplementary Video

Supplementary Movie 3. Live-cell imaging of P21 mRNAs using MCP-GFP-eRF3. U2OS cells were treated with 10 μM Nutlin-3 for 4 h before imaging. To observe the mRNA movements in cytoplasm, MCP-GFP-eRF3 containing a nuclear localization signal was used. The intense signal at the lower part of the movie is the signal from the nucleus. The movie was acquired at 20 frames s−1 in one z plane. Scale bar, 4 µm.

Supplementary Video

Supplementary Movie 4. Live-cell imaging of P21 mRNAs using MCP-GFP-PABPC1*. U2OS cells were treated with 10 μM Nutlin-3 for 4 h before imaging. To observe the mRNA movements in the cytoplasm, MCP-GFP-PABPC1* containing a nuclear localization signal was used. The intense signal on the right side of the movie is the signal from the nucleus. The movie was acquired at 20 frames s−1 in one z plane. Scale bar, 4 µm.

Supplementary Video

Supplementary Movie 5. Live-cell imaging of P21 mRNAs using MCP-GFP. U2OS cells were treated with 10 μM Nutlin-3 for 4 h before imaging. To image mRNA movements in the cytoplasm, MCP-GFP containing a nuclear localization signal was used. The intense signal at the upper-right corner of the movie is the signal from the nucleus. The movie was acquired at 20 frames s−1 in one z plane. Scale bar, 4 µm.

Source data

Source Data Extended Data Fig. 4

Unprocessed western blots of Extended Data Fig. 4

Source Data Extended Data Fig. 10

Unprocessed western blots of Extended Data Fig. 10b

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Li, W., Maekiniemi, A., Sato, H. et al. An improved imaging system that corrects MS2-induced RNA destabilization. Nat Methods 19, 1558–1562 (2022). https://doi.org/10.1038/s41592-022-01658-1

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