β-globin exon 1 is bisected by a sharp border between nonsense codons that do or do not activate NMD. Northern blot analysis of HeLa cells transiently transfected with normal (N) or nonsense mutated β-globin genes with the endogenous 5′ UTR and 3′ UTR (A) or with the 5′ UTR provided by the pCI-neo vector including a 5′ UTR intron (B). Cells were cotransfected with a β-globin gene with extended second and third exons to control for transfection efficiency and loading. The positions of the nonsense mutations are depicted in the schematic representation in part A. The shaded region represents the NMD-resistant area in exon 1. Percentages (%) are the mean of at least three independent experiments. (SEM) Standard error of the mean.

Mutation of methionine 55 restores NMD sensitivity of nonsense mutations in exon 1. (A) Schematic representation of the β-globin gene with nonsense mutations at positions 3, 9, 16, 26, and 39. (Shaded) NMD-resistant 5′ region. Met55 designates the position of the only in-frame methionine codon in the β-globin gene. (B) Northern blot analysis of total cytoplasmic RNA from HeLa cells that were transfected with the expression plasmids for the normal β-globin gene or variants with nonsense mutations at the indicated positions and with or without the Met55Arg mutation. Percentages (%) represent the mean of five independent experiments ±SEM.

Shortening the distance between the initiation codon and the nonsense codon reduces NMD sensitivity by inducing reinitiation of translation. (A) Schematic representation of exons 1 and 2 of the β-globinv gene series of constructs and variants with in-frame deletions of codons 2 through 11 (Δ2-11) and with nonsense mutations at positions 16, 26, or 39. Numbers refer to the position in the undeleted gene. Numbers behind arrows indicate the position of the nonsense mutations with respect to the initiator AUG in the deletion constructs. (B) Northern blot analysis of transcripts expressed in HeLa cells from the β-globinv construct series or the Δ2-11variants. (C) Immunoblot analysis of proteins expressed from the constructs described in A and B.

Read-through does not account for NMD insensitivity in exon 1. (A) Schematic representation of the β-globin gene with nonsense mutations at codons 16 and 39. (Shaded) NMD-resistant 5′ region in the β-globin mRNA. (B) Northern blot analysis of total cytoplasmic RNA from HeLa cells transfected with expression plasmids for the normal β-globin gene or alleles that contain the indicated nonsense mutations. Percentages (%) represent the mean of four independent experiments ±SEM.

Translation reinitiation after termination codons in exon 1. (A) Schematic representation of the β-globinv gene, which contains a venus open reading frame (ORF) fused in-frame to the 3′ end of the β-globin sequence. (Bars) The positions of nonsense codons 3, 9, 12, 16, 20, 26, and 39, as well as of Met55. (Green lines under the scheme) Full-length or N-terminally truncated globinv proteins as expressed from a normal gene or after termination at a nonsense codon and reinitiation at Met55. (B) Fluorescence imaging of HeLa cells that were either transfected with normal or nonsense-mutated β-globinv variants and with a cherry expression plasmid as transfection efficiency control. DAPI staining serves as a reference for image exposure. Scale bar = 20 μm. (C) Immunoblot analysis of cytoplasmic lysates from HeLa cells that were transfected with normal or NS-mutated β-globinv genes (left panel) or variants where the Met55 codon was mutated as described in Figure 3 (right panel). Cotransfection of a YFP plasmid served as control. (Open arrows) The proteins that result from minor reinitiation at unknown non-AUG codons. All proteins were detected by a polyclonal anti-GFP antibody. (D) As C but including the double mutant NS16/39. (Arrow) A protein that probably results from initiation in the normal transcript at Met55 by leaky scanning and that is absent in the Met55Arg variants. (E) Immunoblot analysis of normal or NS9 β-globinv gene expression, or the expression of a β-globinv gene variant where the normal initiation codon has been mutated to a glycine codon (Met_ini→Gly). (F, upper panel) Schematic representation of the positions of the Met55 codon and the three out-of-frame AUG codons (open arrowheads) in exon 2 of the β-globin transcript as well as the position of the TGA87/88 codon in the 3′ NMD-insensitive region (black arrowhead). (Light gray regions) The NMD-resistant areas in exons 1 and 3. (Lower panel) Immunoblot analysis of proteins produced by normal, NS16, or NS39 β-globinv genes that contain a Met55Arg TGA87/88TGC double mutation. Expression was analyzed in the normal translational reading frame, or alternatively in the +1 or the −1 frames of translation. The analysis of expression from the alternative frames was enabled by inserting one or two cytosine residues, respectively, immediately 5′ to the venus ORF.

Increasing the distance between the initiation codon and the nonsense codon does not restore NMD sensitivity when translation reinitiation is maintained. (A) Schematic representation of the 5′ part of the β-globinv gene series of constructs and variants with nonsense mutations at positions 16, 26, or 39 and with in-frame insertions of 10 codons between codons 2 and 3 (ins 2/3) in exon 1. In the ins2/3Δ42-51 series of constructs in addition, codons 42 and 51 were deleted. Numbers behind the arrows indicate the position of the nonsense mutations with respect to the initiator AUG in the ins2/3 and in the ins2/3Δ42-51 constructs. (B) Northern blot analysis of transcripts expressed from the β-globinv construct series or the variants with the ins2/3 insertion and with the additional deletion of codons 42–51. Percentages (%) are means of at least five independent experiments ±SEM. (C) Immunoblot analysis of proteins expressed from the constructs described in A and B.