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

Abstract

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

Figure 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.

Figure 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).

Figure 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.