Figure 3.
5′TOP mRNAs accumulate in SGs during amino acid starvation. (A) RNA-FISH for rpL32-β-globin 5′TOP reporter mRNA (panels 1,4) or wild-type (wt) β-globin mRNA (panels 7,10), transiently expressed in HeLa cells. (Panels 2,5,8,11) Transiently expressed GFP-tagged TIA-1 marks SGs. (Panels 3,6,9,12) Merged images (rpL32-β-globin, red; GFP-TIA-1, green). Panels 1–3 and 7–9 are from HeLa cells cultured in full DMEM medium (Full), whereas panels 4–6 and 10–12 are from HeLa cells under amino acid starvation conditions (−AA). Enlargements of boxed areas are shown at the bottom right of each picture, and the percent of cells with rpL32-β-globin mRNA colocalizing with GFP-TIA-1 in SGs are given in the merged panels, with the number of counted cells in parentheses. (B) Quantification of the fraction of cells in which the rpL32-β-globin 5′TOP reporter mRNA (rpL32) concentrates in SGs over time (0–8 h) of amino acid starvation, compared with wild-type β-globin mRNA (wt) lacking a 5′TOP. Error bars represent standard deviation calculated from three independent experiments (n = 3), in each of which 50 cells were counted. (C) RNA-FISH for endogenous rpL29 (panels 1,4) and GAPDH (panels 7,10) mRNAs in HeLa cells grown in full medium (Full) or under amino acid starvation conditions for 2 h (−AA), as indicated. (Panels 2,5,8,11) Transiently expressed GFP-PABPC1 marks SGs. Merged panels on the right show mRNAs in red and GFP-PABPC1 in green.