Expression analysis of the aberrantly spliced human β-globin transcripts under conditions of enabled and disabled NMD.

(A) RPAs using total RNA of cells transfected with either the normal human β-globin gene (WT, lanes 1 and 2), a gene with the IVS1 + 5 G>A mutation (IVS1 + 5, lanes 3 and 4), or a gene with the IVS1 + 5 G>A mutation and inactivation of the cryptic splice site at position −16 (Δ −16 IVS1 + 5, lanes 5 and 6). NMD was specifically enabled by iron treatment of transfected cells (lanes 1, 3, and 5) or specifically disabled by iron depletion (lanes 2, 4, and 6). Lane 7 represents the analysis of RNA from untransfected HeLa cells. The protected fragments of 205 nts, 187 nts, 175 nts, 159 nts, and 137 nts result from splicing at the normal splice site or the 3 cryptic splice sites, respectively. The 175-nt exon 1 fragment is specific for the normally spliced transcript, is unaffected by activation or inactivation of NMD, and serves as an internal reference for quantification of the aberrant transcripts (lanes 3 and 4) specifically represented by the 187-nt, 159-nt, and 137-nt fragments, respectively. The 205-nt exon 2 fragment reflects the cumulative expression of all 4 mRNA species derived from the parent pre-mRNA. Expression levels of the different species of mature transcripts of the IVS1 + 5 pre-mRNA are indicated in percentages relative to the internal reference (ref). (B) Immunoblot analysis of cells transfected with constructs WT and IVS1 + 5 under conditions of enabled and disabled translation.