Expression analysis of human β-globin constructs with deletions of intron 1.

(A) Schematic representation of the constructs used for transfection. Construct NS39 ΔIVS1 contains a nonsense mutation at position 39 (arrow). In constructs −16 ΔIVS1 and −38 ΔIVS1, intron 1 and 16 nts or 38 nts of the exon 1 sequence are deleted, resulting in a transcript with termination codons at positions 55 (PTC 55*) and 30 (PTC 30*), respectively. (B) Northern blot analysis with a β-globin–specific exon 3 probe (top panel) of total cytoplasmic RNA extracted from cells transfected with constructs NS39 ΔIVS1 (lanes 1 and 2), −16 ΔIVS1 (lanes 3 and 4), −38 ΔIVS1 (lanes 5 and 6), and the normal β-globin construct (WT, lanes 7 and 8) under conditions of enabled and disabled translation. 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 7 and 8) 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. The immunoblot (bottom panel) shows the specific suppression of translation under conditions of iron depletion.