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Optogenetic control of mRNA localization and translation in live cells

Nature Cell Biology volume 22, pages 341–352 (2020)Cite this article

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Abstract

Despite efforts to visualize the spatio–temporal dynamics of single messenger RNAs, the ability to precisely control their function has lagged. This study presents an optogenetic approach for manipulating the localization and translation of specific mRNAs by trapping them in clusters. This clustering greatly amplified reporter signals, enabling endogenous RNA–protein interactions to be clearly visualized in single cells. Functionally, this sequestration reduced the ability of mRNAs to access ribosomes, markedly attenuating protein synthesis. A spatio–temporally resolved analysis indicated that sequestration of endogenous β-actin mRNA attenuated cell motility through the regulation of focal-adhesion dynamics. These results suggest a mechanism highlighting the indispensable role of newly synthesized β-actin protein for efficient cell migration. This platform may be broadly applicable for use in investigating the spatio–temporal activities of specific mRNAs in various biological processes.

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Fig. 1: Design of mRNA-LARIAT.
Fig. 2: Light-induced trapping of specific mRNA molecules in clusters.
Fig. 3: Inhibition of target-mRNA translation by mRNA-LARIAT.
Fig. 4: RCas9-based mRNA-LARIAT targeting of endogenous β-actin mRNA.
Fig. 5: Perturbation of endogenous β-actin mRNA translation and localization during cell migration.
Fig. 6: Monitoring cell morphology under the local or global perturbation of endogenous β-actin mRNA translation.
Fig. 7: Essential and distinct role of newly synthesized β-actin protein in cell migration through the regulation of the focal-adhesion maturation process.

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Data availability

All data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank all of the members of the Heo laboratory for their support and advice. This work was supported by the Institute for Basic Science (grant no. IBS-R001-D1), KAIST Institute for the BioCentury and KBRI basic research program through the Korea Brain Research Institute funded by the Ministry of Science and ICT (grant no. 19-BR-03-02), Republic of Korea.

Author information

Author notes

  1. These authors contributed equally: Na Yeon Kim, Sangkyu Lee.

Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Na Yeon Kim, Jeonghye Yu, Hyerim Park & Won Do Heo

  2. Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, Republic of Korea

    Sangkyu Lee, Nury Kim, Seong Su Won & Won Do Heo

  3. KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Won Do Heo

  4. Cancer Metastasis Control Center, KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Won Do Heo

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Contributions

N.Y.K., S.L. and W.D.H. conceived the project and directed the work. N.Y.K., S.L. and W.D.H. designed the experiments. N.Y.K., S.L., J.Y. and S.S.W. performed the experiments. N.Y.K., S.L., J.Y., N.K., H.P. and W.D.H. discussed the data. N.K. designed the quantification analysis. N.Y.K., S.L. and W.D.H. wrote the manuscript.

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Correspondence to Sangkyu Lee or Won Do Heo.

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Extended data

Extended Data Fig. 1 Trapping of target mRNAs with NLS-MS2-based mRNA-LARIAT.

a, Fluorescence images of HeLa cells co-expressing iRFP-MBS, NLS-MCP–GFP and either VHH(GFP)-CRY2-P2A-CIB1-MP (+VHH(GFP)) or CRY2-P2A-CIB1-MP (–VHH(GFP)) from three independent experiments. Cells were illuminated by blue light at 10-s intervals for 5 min. b, HeLa cells co-expressing VHH(GFP)-CRY2-P2A-CIB1-MP, iRFP-MBS and either NLS-MCP–GFP or NLS-GFP were subjected for fluorescence in situ hybridization targeting the MBS. Blue light was delivered at 5-min intervals for 1 h with an LED array. Co-localization of the FISH probe with NLS-MCP–GFP or NLS-GFP (n = 35 (GFP) and 42 (MCP–GFP) cells). c, Graph showing ratio of iRFP-MBS transcripts trapped in clusters measured by FISH signal. The cluster intensity (Icluster) was divided by the total intensity (Itotal) of the whole cell (n = 35 (GFP) and 88 (MCP–GFP) cells). Data shown as mean ± s.e.m. of three independent experiments in b and c. The statistics in b and c (P values and the error bars) were derived based on n = 3 independent experiments. d, Scatter plot showing relationship between the ratio of trapped MBS transcripts and cluster sizes (n = 3176 clusters from three independent experiments). r is the Pearson’s correlation coefficient. The fluorescence of each cluster was quantified after background signal elimination by measuring median intensity value within each cell boundary for c and d. e, Fluorescence images of HeLa cells demonstrating spatial control of mRNA trapping with mRNA-LARIAT. Light was illuminated at 1-min intervals for 10 min. Yellow circle indicates the region of light stimulation. Dotted lines indicates cell boundaries. White boxes indicate regions for enlarged images. Yellow arrows indicate clusters including MCP signals. Statistical significance was calculated using Student’s two-tailed t-test for b and c. Scale bars, 20 μm. Statistical source data are shown in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Experimental scheme for analysing trapping of ribosomal components with target mRNAs in clusters.

Sequestration of ribosomal components with target mRNAs was tested in various conditions depending on serum or puromycin treatment. Cells were either pre-treated or not treated with puromycin for 4 h. Then all cells were stimulated by blue light with an LED at 3-min intervals for 3 h. Cells with puromycin were either washed before or while light stimulation for 1 h. For serum starvation, cells were starved for 24 h. Then serum was re-added before or while light stimulation for 2 h. Endogenous ribosomes were visualized by staining with antibodies (Ab) against small and large ribosomal subunits or rRNA FISH probes.

Extended Data Fig. 3 Interaction analysis of mRNA and ribosomal components and their restricted dynamics under sequestration of target mRNAs.

a, Representative fluorescence images of HeLa cells co-expressing FusionRed-MBS, MCP–GFP, and VHH(GFP)-CRY2-P2A-CIB1-MP from three independent experiments. Experiments were performed as depicted in Extended Data Fig. 2. rpS6 proteins were visualized by antibody staining. b-e, Graphs showing co-localization of MCP–GFP with b, rpS6 (Top, n = 39 (Blue), 52 (Magenta), 64 (Yellow) and 49 (Green) cells; Bottom, n = 45 (Blue), 48 (Magenta), 46 (Yellow) and 46 (Green) cells); c, RPL10A (Top, n = 44 (Blue), 42 (Magenta), 40 (Yellow) and 40 (Green) cells; Bottom, n = 111 (Blue), 159 (Magenta), 214 (Yellow) and 160 (Green) cells); d, 18S rRNA (Top, n = 40 (Blue), 37 (Magenta), 37 (Yellow) and 42 (Green) cells; Bottom, n = 35 (Blue), 34 (Magenta), 35 (Yellow) and 34 (Green) cells) or e, 28S rRNA (Top, n = 39 (Blue), 37 (Magenta), 37 (Yellow) and 37 (Green) cells; Bottom, n = 35 (Blue), 40 (Magenta), 38 (Yellow) and 38 (Green) cells). HeLa cells for b and c and NIH3T3 cells for d and e were analysed from three independent experiments. f, Representative fluorescence images showing signals of MCP–GFP and rpS6 from three independent experiments. Cells were treated with puromycin for 1 h prior to light stimulation (3-min intervals for 2 h). Or, during light stimulation for 2 h, puromycin was treated at t = 1 h. g, Co-localization of MCP–GFP and rpS6. Data shown as mean ± s.e.m. (n = 41 (Grey) and 43 (yellow) cells pooled from three independent experiments). The statistics in b-e and g (P values and the error bars) were derived based on n = 3 independent experiments. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparisons test for b to e and Student’s two-tailed t-test for g. Scale bars, 20 μm. Statistical source data are shown in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Effects of mRNA tagging and co-expression of RNA-binding proteins on translation.

a, Scatter-plot graphs showing correlation between number of mCherry (mCh) mRNA and intensity of expressed mCherry protein in individual cells. Cells were transfected with expression constructs as indicated. r is the Pearson’s correlation coefficient. n = 71, 59, 52, 60, 57 cells from three independent experiments. b, Graph showing ratio of mCherry intensity to the number of mRNA for each group of cell. Statistical source data are shown in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Dependency of cluster formation on the amount of target transcript.

a, Levels of mCherry-MBS mRNA relative to those of GAPDH, determined by qRT-PCR, after treatment of different concentrations of doxycycline for 24 h (n = 3 independent experiments were used to derive statistics). b, Representative fluorescence images showing clusters, translated mCherry protein and MBS transcript (detected by FISH probe) under doxycycline treatment and light stimulation from three independent experiments. Scale bars, 20 μm. c, Average size and d, the number of clusters under treatment of different concentrations of doxycycline. Data shown as mean ± s.e.m., the statistics in c and d (P values and the error bars) were derived based on n = 48 (Magenta), 41 (Yellow), 44 (Green) and 43 (Blue) cells pooled from three independent experiments. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparisons test for a, c and d. Statistical source data are shown in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Effects of blue light illumination and cluster formation on cell physiology.

a, Immunoblots detecting levels of p-eIF2α and eIF2α in the absence or presence of light (10-s light pulses delivered at 5-min intervals for 24 h with an LED array) and with or without 0.2 μM thapsigargin treatment to NIH3T3 cells. b, Normalized level of p-eIFα by total eIF2α level. n = 3 independent experiments. Data shown as mean ± s.e.m.. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparisons test. c, Representative fluorescence images showing cluster formation and reversible assembly of stress granules. NIH3T3 cells expressing MS2-based mRNA-LARIAT components and FusionRed(FuRed)-G3BP1 were illuminated by light at 20-s interval for 3 h. After 1-h illumination, 200 μM sodium arsenite (NaAsO2) was treated for 1 h. Then, 25 μg ml−1 cycloheximide (CHX) was treated for another 1 h. d, Time-lapse images showing assembly of stress granules by NaAsO2 treatment in the presence of clusters. Three independent experiments were performed for c and d. Scale bars, 20 μm. Statistical source data and unprocessed blots are shown in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 Specificity and sensitivity of RCas9-based mRNA-LARIAT.

a, Representative fluorescence images showing HeLa cell co-expressing dCas9-GFP, sgRNA and PAMmer targeting GAPDH and LARIAT components (CRY2-P2A-CIB1-MP (–VHH(GFP)) or VHH(GFP)-CRY2-P2A-CIB1-MP (+VHH(GFP))). Cells were illuminated with light for 10-s at 5-min intervals for 1 h with an LED array. GAPDH mRNAs were visualized with a Quasar 670-labelled FISH probe. b, Co-localization analysis of cluster signals with GAPDH FISH probe (n = 38 (Magenta), 39 (Yellow), 51 (Green) and 61 (Blue) cells). Statistical significance was calculated using one-way ANOVA with Tukey multiple comparisons test. Data shown as mean ± s.e.m. and the statistics (P values and the error bars) were derived based on n = 3 independent experiments. c, Fluorescence images of HeLa cells co-expressing dCas9-GFP, β-actin sgRNA, β-actin PAMmer and VHH(GFP)-LARIAT (VHH(GFP)-FusionRed-CRY2-P2A-CIB1-MP). Cells were stained with FISH probes against transcripts as indicated. d, Fluorescence images of NIH3T3 cells co-expressing RCas9-based mRNA-LARIAT components targeting Arp2 or Arp3 mRNA. Scale bars, 20 μm. Data are representative of three independent experiments for a-d. Statistical source data are shown in Source Data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 Trapping of target mRNAs with NLS-RCas9-based mRNA-LARIAT.

a, Representative fluorescence images of HeLa cells co-expressing NLS-dCas9-GFP, sgRNA and PAMmer against β-actin and LARIAT components (VHH(GFP)-CRY2-P2A-CIB1-MP (+VHH(GFP)) or CRY2-P2A-CIB1-MP (–VHH(GFP))). Cells were illuminated by blue light at 10-s intervals for 5 min. b, HeLa cells co-expressing VHH(GFP)-CRY2-P2A-CIB1-MP and NLS-RCas9 components against β-actin or NLS-dCas9-GFP only were subjected for FISH targeting the β-actin after blue light stimulation. Co-localization of the β-actin FISH probe with NLS-RCas9-GFP or NLS-dCas9-GFP (n = 27 (dCas9 only) and 82 (RCas9) cells). c, Graph showing ratio of β-actin mRNAs trapped in clusters measured by FISH signal. The cluster intensity (Icluster) was divided by the total intensity (Itotal) of the whole cell (n = 23 (dCas9 only) and 68 (RCas9) cells). d, Scatter plot showing relationship between the ratio of trapped β-actin mRNAs and cluster sizes (n = 1426 clusters). r is the Pearson’s correlation coefficient. Blue light was delivered at 5-min intervals for 1 h with an LED array. The fluorescence of each cluster was quantified after background signal elimination by measuring median intensity value within each cell boundary for c and d. Statistical significance was calculated using Student’s two-tailed t-test. Scale bars, 20 μm. Data in b and c are shown as mean ± s.e.m. and the statistics (P values and the error bars) were derived based on n = 3 independent experiments. Data are representative of three independent experiments for a-d. Statistical source data are shown in Source Data Extended Data Fig. 8.

Source data

Extended Data Fig. 9 Interaction analysis of mRNA and ribosomal components and their restricted bindings under sequestration of target mRNAs.

a, Representative fluorescence images of HeLa cells co-expressing VHH(GFP)-CRY2-P2A-CIB1-MP and RCas9 components against β-actin mRNA. Experiments were done as depicted in Extended Data Fig. 2. Endogenous rpS6 proteins were visualized by antibody staining. Scale bars, 20 μm. b-e, Graphs showing co-localization of RCas9-GFP with b, rpS6 (Top, n = 41 (Blue), 57 (Magenta), 53 (Yellow) and 44 (Green) cells; Bottom, n = 47 (Blue), 46 (Magenta), 48 (Yellow) and 46 (Green) cells); c, RPL10A (Top, n = 42 (Blue), 49 (Magenta), 46 (Yellow) and 54 (Green) cells; Bottom, n = 160 (Blue), 214 (Magenta), 159 (Yellow) and 111 (Green) cells); d, 18S rRNA or e, 28S rRNA. For top graphs on d and e, n = 34 (Blue), 35 (Magenta), 33 (Yellow) and 34 (Green) cells for puromycin treatment analysis; For bottom graphs on d and e, n = 40 (Blue), 38 (Magenta), 38 (Yellow) and 37 (Green) cells. HeLa cells for b and c and NIH3T3 cells for d and e were experimented as depicted in Extended Data Fig. 2. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparisons test. Data in b-e are shown as mean ± s.e.m. and the statistics (P values and the error bars) were derived based on n = 3 independent experiments. Data are representative of three independent experiments for a-e. Statistical source data are shown in Source Data Extended Data Fig. 9.

Source data

Extended Data Fig. 10 Changes of subcellular distribution of endogenous β-actin mRNAs induced by mRNA-LARIAT.

(a) Polarization index was quantified with β-actin FISH intensities. (b) Dispersion index was quantified with β-actin FISH intensities. (c) Scatter plot of distribution and polarity index of β-actin mRNA. n = 53 (Black), 59 (Grey), 64 (Magenta) and 105 (Blue) cells from three independent experiments. r is the Pearson’s correlation coefficient. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparisons test. Data in a and b are shown as mean ± s.e.m. and the statistics (P values and the error bars) were derived based on n = 3 independent experiments. Statistical source data are shown in Source Data Extended Data Fig. 10.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Live-cell imaging of HeLa cell showing reversible cluster formation by blue-light illumination based on the MS2-based mRNA-LARIAT system. Images were captured every 20 s with one pulse stimulation. The experiment was repeated three times independently with similar results.

Supplementary Video 2

Live-cell imaging of a HeLa cell forming blue-light-mediated clusters using the MS2-based mRNA-LARIAT system. Images were captured every 10 s. The experiment was repeated three times independently with similar results.

Supplementary Video 3

Live-cell imaging of a cultured hippocampal neuron forming light-induced clusters. Images were captured every 20 s. The experiment was repeated three times independently with similar results.

Supplementary Video 4

Live cell imaging of HeLa cells forming light-induced clusters using the RCas9-based mRNA-LARIAT system. Images were captured every 10 s. The experiment was repeated three times independently with similar results.

Supplementary Video 5

Live-cell imaging of HeLa cell proliferation under protein clustering. Images were captured every 3 min. The experiment was repeated three times independently with similar results.

Supplementary Video 6

Live-cell imaging of a NIH3T3 cell migrating under conditions of light-induced sequestration of endogenous β-actin mRNAs. Images were captured every 5 min. The experiment was repeated six times independently with similar results.

Supplementary Video 7

Live-cell imaging of a NIH3T3 cell locally illuminated with light on the leading edge. Images were captured every 2 min. The experiment was repeated three times independently with similar results.

Supplementary Video 8

Live-cell imaging of NIH3T3 cells changing protrusion activity when β-actin mRNAs were locally trapped. Images were captured every 2 min. The experiment was repeated three times independently with similar results.

Supplementary Video 9

Live-cell imaging of NIH3T3 cells morphologies under the indicated chemicals and/or light stimulation. Images were captured every 5 min. The experiment was repeated four times independently with similar results.

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Kim, N.Y., Lee, S., Yu, J. et al. Optogenetic control of mRNA localization and translation in live cells. Nat Cell Biol 22, 341–352 (2020). https://doi.org/10.1038/s41556-020-0468-1

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