Internal ribosome entry sequence-mediated translation initiation triggers nonsense-mediated decay

Abstract

In eukaryotes, a surveillance pathway known as nonsense-mediated decay (NMD) regulates the abundance of messenger RNAs containing premature termination codons (PTCs). In mammalian cells, it has been asserted that the NMD-relevant first round of translation is special and involves initiation by a specific protein heterodimer, the nuclear cap-binding complex (CBC). Arguing against a requirement for CBC-mediated translation initiation, we show that ribosomal recruitment by the internal ribosomal entry sequence of the encephalomyocarditis virus triggers NMD of a PTC-containing transcript under conditions in which ribosome entry from the cap is prohibited. These data generalize the previous model and suggest that translation per se, irrespective of how it is initiated, can mediate NMD.

Figure 1

Constructs used in this study. Construct (A) contains a stem–loop structure to prevent ribosomal read-through, the EMCV IRES to provide internal translation initiation and the β-globin gene as a reporter for NMD. Construct (B) was used as a control for cap-dependent translation, and contains the β-globin gene as an NMD reporter. Construct (C) contains the same features as construct (A), except that the IRES is mutated. Bicistronic construct (D) contains a Renilla luciferase reporter for cap-dependent translation and a firefly luciferase reporter for IRES-dependent translation. Bicistronic construct (E) contains the same features as construct (D), except that the IRES is mutated. EMCV, encephalomyocarditis virus; IRES, internal ribosome entry sequence; NMD, nonsense-mediated decay.

Figure 2

NMD is supported by cap-dependent and IRES-dependent translation. (A) Representative northern blot of cap-dependent β-globin WT and NS constructs (lanes 1–4) and IRES WT and NS constructs (lanes 4–8). NS-containing constructs in both cases were present at 10–30% of the level of the WT transcript (lanes 2 and 6) in control cells treated with siRNA against β-gal. siRNA directed against central NMD factor UPF1 increased levels of NS messenger RNA three- to fourfold (lanes 4 and 8). The control mRNAs are extended β-globin forms (see Methods). The percentages shown are mean values and standard deviations (s.d.) derived from the results of at least three independent experiments. (B) Immunoblotting demonstrates the efficacy of RNA interference against UPF1. The level of UPF1 protein in cells treated with siRNA against UPF1 (lane 1) is less than 13% of that in control cells treated with siRNA against β-gal, as shown by the dilution series in lanes 2–6. β-gal, β-galactosidase; IRES, internal ribosome entry sequence; NMD, nonsense-mediated decay; NS, nonsense mutation; siRNA, short interfering RNA; WT, wild type.

Figure 3

IRES-dependent translation is specifically abolished by an IRES point mutation. (A) Immunoblotting for the β-globin protein. The upper band corresponds to the extended β-globin product from the WT+300+e3 transfection control and the lower band corresponds to β-globin protein produced from the cap WT construct (lane 1), the mut IRES WT construct (lane 2), the IRES WT construct (lane 3) or empty vector (lane 4). (B) Luminometer measurements of firefly luciferase produced from the bicistronic luciferase constructs shown in Fig 1D,E, after normalization to Renilla luciferase activity. Ctrl, control; IRES, internal ribosome entry sequence; mut, mutant; WT, wild type.

Figure 4

The mut IRES construct does not support NMD. A representative northern blot of IRES WT and NS constructs (lanes 1–4) and mut IRES WT and NS constructs (lanes 4–8) is shown. For the intact IRES, in control cells treated with β-gal siRNA, levels of the NS construct were 10–25% of levels of the WT construct (lanes 3 and 4). The NS construct increased three- to fourfold in cells treated with UPF1 siRNA (compare lanes 2 and 4). In control cells treated with β-gal siRNA, in contrast, the mutant IRES NS construct was present at 65–100% of the level of the mutant IRES WT construct (lanes 7 and 8), and in cells treated with UPF1 siRNA, the abundance of the mutant IRES NS construct failed to increase (compare lanes 6 and 8). The percentages shown are mean values and standard deviations (s.d.) derived from the results of at least three independent experiments. β-gal, β-galactosidase; IRES, internal ribosome entry sequence; mut, mutant; NMD, nonsense-mediated decay; NS, nonsense mutation; siRNA, short interfering RNA; WT, wild type.