Figure 3. PTC+ mRNAs Do Not Have Processing Defects.

(a) Equal amount of WT and 39T β-globin DNA constructs were transfected into HeLa cells, 12, 24 or 48 hrs later, FISH of β-globin transcripts was carried out. At 12 hr post-transfection, in 84 ± 5% of cells transfected with WT β-globin plasmid, the mRNA was mainly cytoplasmic, and in 76 ± 7% of cells transfected with 39T β-globin plasmid, the mRNA was mainly nuclear. (b) Schematic of intron-containing and intron-lacking β-globin constructs are shown and the size (in nts) of the introns and exons are indicated. Primers are indicated by the arrows. The GInExR primer is complementary to the junction of intron2-exon3 and the GExExR primer is complementary to the junction of exon2-exon3. Equal amount of WT and 39T β-globin constructs (intron-containing) were transfected into HeLa cells and 4 hrs later, α-amanitin was added. Total RNA was extracted at 0 hr, 4 hr and 8 hr after addition of α-amanitin, followed by RT-PCR using the two sets of primers as indicated separately. Equal volume of PCR products were mixed and loaded on the gel. The PCR products amplified from intron-containing and intron-lacking plasmid DNAs were used as size markers of pre-mRNA and mRNA respectively. The sizes (in kb) of the DNA marker are indicated. (c) Same as (b), except that Smad DNA constructs were transfected and PCR was carried out using primers as indicated. See also Figure S4.