Expression analysis of −38 ΔIVS1 constructs with inactivation of downstream AUG codons.

(A) The 4 downstream AUG codons that may be used as translation-reinitiation sites in the −38 transcript are indicated by arrows. (B) Northern blot analysis with a β-globin–specific exon 3 probe of the normal construct (WT) and the −38 ΔIVS1 constructs that lack either all 4 downstream AUG codons (ΔAUG 1-4, lanes 1 and 2) or single AUG codons (ΔAUG 1, lanes 3 and 4; ΔAUG 2, lanes 5 and 6; ΔAUG 3, lanes 7 and 8; and ΔAUG 4, lanes 9 and 10). Translation and NMD were regulated specifically by iron depletion and repletion. The WT-ΔIVS1-CAT plasmid was cotransfected to control for transfection efficiency. The ratio of WT-RNA expression under conditions of active and inactive translation (lanes 11 and 12) was used to control for an unspecific translation dependent slight increase in mRNA expression. Values are the mean results from 3 independent experiments after normalization for transfection efficiency and after considering the unspecific (∼ 20%) reduction in RNA expression under conditions of translation inhibition.