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. 2024 Feb 16;10(7):eadi7830. doi: 10.1126/sciadv.adi7830

Fig. 1. Single-molecule imaging recapitulates 5′TOP translational repression.

Fig. 1.

(A) Schematic representation of reporter mRNAs for single-molecule imaging of translation. The 5′TOP reporter contains the full-length RPL32 5′UTR, whereas the canonical reporter has a control 5′UTR of similar length. Black arrows indicate transcription start sites. (B) Representative images of canonical and 5′TOP reporter mRNAs (MCP-Halo foci, magenta) undergoing translation (scFv-GFP foci, green) in the absence or presence of mTOR inhibitor Torin 1 (250 nM, 1 hour). Scale bars, 5 μm. (C) Translation site intensities of canonical and 5′TOP reporter mRNAs quantified in absence or presence of Torin 1 (250 nM, 1 hour). SunTag intensities are plotted for all mRNAs (colored circles) overlaid with the mean ± SD (≥1089 mRNAs per condition, n = 3). (D) Fraction of mRNAs undergoing translation quantified per cell for canonical and 5′TOP reporter in the absence or presence of Torin 1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥162 cells per condition, n = 3). For statistics, unpaired t tests were performed, with statistical significance claimed when P < 0.05 (ns, not significant; *P < 0.05; ***P < 0.001).