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.