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. 2023 Jan 8;12(2):259.
doi: 10.3390/cells12020259.

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress

Desislava S Makeeva  1 , Claire L Riggs  2 , Anton V Burakov  1 , Pavel A Ivanov  1 , Artem S Kushchenko  1   3 , Dmitri A Bykov  4   5 , Vladimir I Popenko  5 , Vladimir S Prassolov  5 , Pavel V Ivanov  2 , Sergey E Dmitriev  1   3   5
Affiliations

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress

Desislava S Makeeva et al. Cells. .

Abstract

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

Keywords: ETF1; GSPT1; MCTS1; ligatin; oxidative stress; ribosome recycling; stress granules; translation reinitiation; translation termination; upstream open reading frames uORFs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The rates of translation reinitiation and stop-codon readthrough are increased under conditions of arsenite-induced stress, as revealed by luciferase reporter assays. (A–C) Schematic representation of reporter mRNA constructs (upper panels) and relative luciferase activities in arsenite-treated HEK293T cells transfected with the constructs, normalized to that in untreated cells (lower panels). Reporter mRNAs have either a short uORF (A) or the full-length Rluc-encoding ORF (B) in front of Fluc CDS or harbor a PTC (premature termination codon) in the main (Nluc) CDS (C), as described in the text. In (A) and (C), transcripts encoding Rluc or Fluc, as indicated, were co-transfected as controls; 2 h after transfection, cells were lysed, and reporter-dependent luciferase activities were measured. (D) Translation efficiencies of the reporter mRNAs, divided into those of the corresponding control transcripts after normalization, in untreated or 150 µM arsenite-treated cells are shown (calculated from the same data as in (a–c)). The mean values (±SD) of at least three independent experiments are shown. The absolute values of luciferase units for untreated cells were ~9 × 105 for control Fluc, ~2 ×105 for REI uORF-Fluc, ~3 × 106 for Rluc, ~4 × 105 for control Rluc-Fluc (Fluc), ~3 × 103 for REI Rluc-Fluc (Fluc), ~1 × 107 for Nluc, ~4 × 104 for both PTC-Nluc mRNAs, ~8 × 105 for Fluc; the background (no mRNA) values were 50 units for Fluc and 120 units for Rluc and Nluc.
Figure 2
Figure 2
Translation termination factors are recruited to arsenite-induced SGs. HeLa cells were treated with 250 µM sodium arsenite for 1 h (left panel) or left untreated (right panel), followed by fixation and immunostaining for eRF1 or eRF3 (red false color), as indicated, and an SG marker G3BP1 (green channel). A few representative cells are shown; boxes indicate magnified areas. All large images are of the same magnification (scale bar: 10 μm).
Figure 3
Figure 3
Ribosome recycling factors are recruited to arsenite-induced SGs. HeLa cells were treated with 250 µM sodium arsenite for 1 h (left panel), followed by fixation and immunostaining for the protein of interest along with an SG marker G3BP1. Immunostaining of non-treated cells is presented in the right panel. A few representative cells are shown; boxes indicate magnified areas. All large images are of the same magnification (scale bar: 10 μm).
Figure 4
Figure 4
Changes of translation reinitiation and termination in arsenite-treated wt and ddG3BP1/2 U2OS cells. Wt (A) and ddG3BP1/2 (B) U2OS cells were transfected with the same reporter mRNAs as in Figure 1. Two hours after transfection, cells were lysed, and luciferase activities were measured and normalized, as described above. Translation efficiencies of the reporter mRNAs were normalized by dividing by the translation efficiencies of the corresponding control transcripts, in untreated or 150 µM arsenite-treated cells. The mean values (±SD) of at least three independent experiments are shown. The absolute values were roughly similar to those indicated in the legend in Figure 1 (and were at least two orders of magnitude above the background).
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This research was funded by the Russian Science Foundation, grant RSF 18-14-00291 to S.E.D.

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