Contrary to the transcript corresponding to the minigene lacking intron 6, normal and nonsense-mutated human <i>HFE</i> transcripts are committed to NMD.

<p>(A) Schematic representation of the studied human <i>HFE</i> minigenes: normal (WT), nonsense-mutated (Y138X), lacking intron six (Del_IVS6_WT) and nonsense-mutated lacking intron six minigene (Del_IVS6_Y138X). The position of the initiation (ATG) and termination (STOP) codons is represented. The name of each minigene is indicated above each diagram. (B) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or a control siRNA target (siRNA Luciferase). Twenty-four hours after siRNA treatment, cells were co-transfected with the plasmids encoding the above referred minigenes and with a second dose of siRNAs (UPF or Luciferase). Twenty-four hours later, cells were harvested for protein and RNA. Immunoblotting was performed using a human UPF1 specific antibody and an α-tubulin specific antibody to control for variations in protein loading. (C) <i>HFE</i> mRNA quantification was performed by RT-qPCR using primers specific for the heterologous 5′ UTR common to all transfected genes. Neomycin resistance transcript was used as a normalization control. Quantification of the transcript levels was performed by the absolute quantification method. Levels of <i>HFE</i> mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's <i>t</i> test (unpaired, two tailed).