The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations.

<p>(A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m⁷G) and poly(A) tail [(A)_n] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038505#s4" target="_blank">Materials and Methods</a>). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's <i>t</i> test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in (<i>A</i>). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038505#s4" target="_blank">Materials and Methods</a>). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in (<i>B</i>). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (<i>C</i>). Statistical analysis was performed using Student's <i>t</i> test (unpaired, two-tailed).