Report

An RNA biosensor for imaging the first round of translation from single cells to living animals

See allHide authors and affiliations

Science  20 Mar 2015:
Vol. 347, Issue 6228, pp. 1367-1671
DOI: 10.1126/science.aaa3380
  • Fig. 1 Imaging translation of mRNAs in living cells.

    (A) Schematic of TRICK assay. (B) Schematic of TRICK reporter transcript. 6xPP7 stem-loops (PBS) inserted in-frame with the C terminus of a protein-coding sequence and 24xMS2 stem-loops (MBS) in the 3′ UTR. (C) Expression of TRICK reporter mRNA in U-2 OS cells. The protein encoded by the TRICK reporter (51.4 kD) is translated in U-2 OS cells, and expression is not affected by NLS-MCP-RFP and NLS-PCP-GFP. (D) U-2 OS cell expressing TRICK reporter. Arrows indicate untranslated nuclear mRNA and three untranslated mRNAs detected in the cytoplasm. Scale bar, 10 μm. (E) Cytoplasmic region of untreated U-2 OS cells. (F) Addition of cycloheximide (100 μg ml−1) and (G) addition of puromycin (100 μg ml−1) during ponA induction of TRICK reporter mRNAs. Scale bar (E to G), 2 μm. (H) Percentage of untranslated TRICK mRNAs in U-2 OS cells. In untreated cells, 5.8 ± 1.4% of mRNAs colocalize with both NLS-PCP-GFP and NLS-MCP-RFP compared to 91.0 ± 3.0% for cycloheximide-treated and 92.6 ± 1.0% for puromycin-treated cells. n = 5 cells for each condition.

  • Fig. 2 P-bodies are sites of translation regulation during stress in HeLa cells.

    (A and B) IF-FISH of cells expressing Δ5′ TOP TRICK reporter mRNA [(A), gray] or 5′ TOP TRICK reporter mRNA [(B), gray] during arsenite stress (0.5 mM) contain stress granules (TIAR, green) and P-bodies (DDX6, red). Arrows: mRNA clusters in P-bodies. (C) Fraction of cytoplasmic Δ5′ TOP (n = 19 cells) and 5′ TOP (n =17 cells) mRNAs located within P-bodies after 60 min of arsenite (0.5 mM) stress (P = 0.0009, unpaired t test). (D and E) Live-cell image of 5′ TOP TRICK reporter mRNA during arsenite stress (D) and relief of stress (E). In stressed cells, mRNAs (red, green) in cytosol and P-bodies (cyan) are untranslated. In relieved cells, many mRNAs (red, green) in cytosol have been translated whereas mRNAs retained in P-bodies (cyan) remain untranslated. Arrow: clustered mRNAs. Scale bar (A, B, D, E), 10 μm. (F) Percentage of untranslated mRNAs (cytosol and P-bodies) during stress (n = 9 cells) and relief of stress (n = 10 cells). Upon relief of stress, 5′ TOP mRNAs in P-bodies are not translated (P = 0.31, unpaired t test); mRNAs in the cytosol have undergone translation (P < 0.0001, unpaired t test).

  • Fig. 3 Stage specific translational activation of osk mRNA in Drosophila oocytes.

    (A) 12xPP7 stem-loops in open reading frame of osk mRNA did not inhibit translation in Drosophila oocytes. Both long and short isoforms of wild-type Oskar protein, and Oskar-TRICK fusion protein were detected in ovary extracts. (B) osk-TRICK mRNA monitors stage-specific translational activation in oocytes. Immunodetection of Oskar protein (blue) in egg-chambers simultaneously expressing osk-TRICK mRNA, NLS-PCP-GFP, and NLS-MCP-RFP. Boxed areas: stage 7 oocyte (left) and pole plasm area of a stage 10 oocyte (right). (C) Reduction in NLS-PCP-GFP correlates with Oskar protein abundance and oocyte size. Quantification of fluorescent signals from NLS-PCP-GFP per NLS-MCP-RFP and Oskar protein at the pole plasm of individual oocytes. The size and color of dots indicate the measured area of the oocyte. Pearson correlation (r), linear regression (blue line), and 95% confidence area (gray). Scale bar, 50 μm.

Supplementary Materials

  • An RNA biosensor for imaging the first round of translation from single cells to living animals

    James M. Halstead, Timothée Lionnet, Johannes H. Wilbertz, Frank Wippich, Anne Ephrussi, Robert H. Singer, Jeffrey A. Chao

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S12
    • Captions for Movies S1 to S7
    • References

    Images, Video, and Other Other Media

    Movie S1
    Diffusion of mRNA molecules in the cytoplasm. mRNAs were simultaneously imaged in both channels (NLS-PCP-GFP, green and NLS-MCP-TagRFP-T, red). Most mRNAs appear as red particles. A few colocalized particles can be observed. Frame rate: 15ms/frame.
    Movie S2
    Diffusion of mRNA molecules in the cytoplasm in the presence of CHX (100μg ml-1). mRNAs were simultaneously imaged in both channels (NLS-PCP-GFP, green and NLS-MCP-TagRFP-T, red). Most mRNAs appear as colocalized green/red particles. Frame rate: 15ms/frame.
    Movie S3
    Diffusion of mRNA molecules in the cytoplasm in the presence of Puromycin (100μg ml-1). mRNAs were simultaneously imaged in both channels (NLS-PCP-GFP, green and NLS-MCP-TagRFP-T, red). Most mRNAs appear as colocalized green/red particles. Frame rate: 15ms/frame.
    Movie S4
    Diffusion of mRNA molecules in the nucleus. mRNAs were simultaneously imaged in both channels (NLS-PCP-GFP, green and NLS-MCP-TagRFP-T, red). Most mRNAs appear as colocalized green/red particles. Frame rate: 15ms/frame.
    Movie S5
    Diffusion of mRNA molecules in the nucleus in the presence of low CHX (1μg ml-1). mRNAs were simultaneously imaged in both channels (NLS-PCP-GFP, green and NLS-MCP-TagRFP-T, red). Most mRNAs appear as colocalized green/red particles. Frame rate: 15ms/frame.
    Movie S6
    Movement of mRNAs molecules in the cytoplasm of arsenite (0.5mM) stressed cells. mRNAs were simultaneously imaged in red (NLS-MCP-Halo, Janelia Fluor 549) and green (NLS-PCP-GFP) channels. P-bodies were imaged in cyan (DDX6-Turquoise2) both before and after mRNA imaging to ensure that they did not move during acquisition of mRNA images. Frame rate: 50ms/frame.
    Movie S7
    Movement of mRNAs molecules in the cytoplasm of cells relieved from arsenite stress. mRNAs were simultaneously imaged in red (NLS-MCP-Halo, Janelia Fluor 549) and green (NLS-PCP-GFP) channels. P-bodies were imaged in cyan (DDX6-Turquoise2) both before and after mRNA imaging to ensure that they did not move during acquisition of mRNA images. Frame rate: 50ms/frame.

Stay Connected to Science


Editor's Blog

Navigate This Article