Downregulating Upf1, Upf2, Upf3X or CBP80/20 inhibits nonsense-mediated messenger RNA decay that results from encephalomyocarditis virus internal ribosome entry site-initiated translation. (A) Diagrams of plasmid DNAs. Boxes represent exons, intervening lines signify introns and hp refers to the hairpin structure that blocks translation initiation upstream from the EMCV IRES. ATG and TAA denote translation initiation and termination codons, respectively, and 39 indicates the position of the premature termination codon when present. (B) (upper panel) Western blot analysis (WB) using the specified antibody (anti-) after treatment with Upf1 or control siRNA. The level of p62 was used to control for variations in protein loading. The three leftmost lanes show twofold dilutions of protein, indicating that the analysis was semiquantitative. (lower panel) Reverse transcription–PCR after treatment with Upf1 siRNA or control siRNA. The level of each RLuc-Gl test mRNA was normalized to the level of MUP mRNA. Normalized values were then calculated as a percentage of the normalized value of either RLuc-Gl Norm mRNA or hp-EMCV IRES-RLuc Norm mRNA, each of which was defined as 100%. Values are derived from three independently performed experiments. The five leftmost lanes show twofold dilutions of RNA. (C) As in (B), except that Upf2 siRNA was used instead of Upf1 siRNA. (D) As in (B), except that Upf3X siRNA was used and the level of pb2 was used to control for variations in protein loading as p62 migrates with Upf3X. (E) As in (B), except that CBP80 and CBP20 (CBC) siRNAs were used. CBP20 runs as a smeared band. EMCV, encephalomyocarditis virus; Gl, β-globin; IRES, internal ribosome entry site; Luc, luciferase; MUP, major urinary protein; Norm, PTC-free; R, Renilla; siRNA, short interfering RNA; Ter, PTC-containing.

Nonsense-mediated messenger RNA decay that results from encephalomyocarditis virus internal ribosome entry site-initiated translation reduces the abundance of CBP80-bound mRNA and its eIF4E-bound product to a comparable percentage of the corresponding normal messenger ribonucleoprotein. (A,B, upper panels) Western blot analysis (WB) indicated that the immunoprecipitation (IP) efficiency of CBP80 or eIF4E was 18±4% or 14±2%, respectively—1/100 of each sample was analysed before IP and 1/20 of each sample was analysed after IP. (A,B, lower panels) Reverse transcription–PCR of Gl and MUP mRNAs. Values are derived from two independently performed experiments. EMCV, encephalomyocarditis virus; Gl, β-globin; IRES, internal ribosome entry site; Luc, luciferase; MUP, major urinary protein; Norm, PTC-free; NRS, normal rabbit serum; R, Renilla; Ter, PTC-containing.

4E-BP1 expression fails to inhibit nonsense-mediated messenger RNA decay that results from encephalomyocarditis virus internal ribosome entry site-initiated translation. (A) Western blot analysis (WB) shows that 4E-BP1 expression inhibited the bulk of cellular translation, as measured by the reduced level of the secreted MUP. The level of p62 was used to control for variations in protein loading. The four leftmost lanes show twofold dilutions of protein. (B) RLuc activity assays also indicated that 4E-BP1 expression inhibited the bulk of cellular translation. RLuc activity was normalized to the level of the corresponding RLuc mRNA. (C) Reverse transcription–PCR as in Fig 1B (lower panel). Values are derived from three independently performed experiments. EMCV, encephalomyocarditis virus; Gl, β-globin; IRES, internal ribosome entry site; Luc, luciferase; MUP, major urinary protein; Norm, PTC-free; R, Renilla; Ter, PTC-containing.