Figure 4.

Prolonged retention of PTC+mRNAs in the nucleus requires a proper reading frame. (a) Schematic of β-globin constructs. The black boxes indicate the codons that have been changed. For 39T β-globin, the codon at 39 was changed from CAG to TAG, and for missense mutation at codon 39 (39M), CAG was changed to AAG. For 39T-OutF, the frame was shifted up by deleting a T, which is 6 nt (nucleotide) upstream of TAG, and restored by addition of a C, which is 22 nt downstream of the TAG. For 59T-InF, the frame of the WT β-globin was shifted to create a nonsense mutation at 59th codon by adding a C into 19 nt upstream of the TAA, and the frame was corrected by deleting the A into 18 nt downstream of the 59th TAA. (b) PCR products (100 ng μl⁻¹) amplified from β-globin constructs using CMV-F and pCDpAR primers were microinjected into nuclei of HeLa cells, and α-amanitin was added 30 mins after microinjection. fluorescence in situ hybridization (FISH) of β-globin transcripts was carried out at 1 h after addition of α-amanitin. Insets show FITC-conjugated dextran coinjected as an injection marker. The graph shows the average N/C ratios for β-globin mRNAs, and error bars indicate the standard errors among three independent experiments. Statistical analysis was performed as in Figure 1c. (c) The 39th, 122nd, and 133rd nonsense codon was replaced to create missense mutation, respectively. Smad plasmids (50 ng μl⁻¹) were microinjected into the nuclei of HeLa cells, and α-amanitin was added 15 mins after microinjection. FISH of Smad transcripts was carried out at 1 h after addition of α-amanitin. Insets show FITC-conjugated dextran co-microinjected as an injection marker. The graph shows the average N/C ratios for Smad mRNAs, and error bars indicate the standard errors among three independent experiments. Statistical analysis was performed as in Figure 1c.