Mode of transcription of PTC-containing β-globin gene affects nonsense-mediated decay for the transcript. (A) Steady-state pool of cytoplasmic BBB^PTC transcript decays at a rate identical to that of the normal BBB mRNA after being constitutively expressed. The Tet-regulated promoter-driven BBB and BBB^PTC constructs were transiently transfected into NIH 3T3 B₂A₂ cells cultured in the absence of tetracycline to allow constitutive expression of BBB and BBB^PTC transcripts following transfection. Thirty-six hours later, mRNA stability was determined by blocking ongoing transcription with tetracycline. RNA isolation, data quantitation, and plotting were as described in the legend to Fig. 1. Left, Northern blots showing decay of the cytoplasmic steady-state pool of BBB (wild-type) or BBB^PTC (PTC) mRNA constitutively transcribed from the Tet-regulated promoter in proliferating NIH 3T3 B₂A₂ cells. Right, comparison of decay rates of BBB and BBB^PTC mRNAs. (B) Northern blots showing the direct side-by-side comparison of the levels of wild-type (wt) BBB and BBB^PTC transcripts transcribed from the Tet-regulated promoter in a transient (left) or constitutive (right) mode. Transfection, RNA isolation, and data quantitation were as described in Materials and Methods and in the legend to Fig. 1.

Ectopic expression of a dominant-negative mutant of human UPF1 protein slows down deadenylation of PTC-containing β-globin mRNA, leading to message stabilization. (A) Northern blots showing deadenylation and decay of BBB^PTC mRNA in proliferating NIH 3T3 cells in the absence (control) or presence of ectopically expressed wild-type (wt) or R844C mutant (mt) hUpf1 protein. (B) Comparison of deadenylation and decay rates of BBB^PTC mRNA in the absence (control) or presence of ectopically expressed wt Upf1 or mt Upf1 protein. Transient transfection, time course experiments, RNA isolation, poly(A)⁻ RNA preparation, poly(A) length comparisons, and data quantitation and plotting were as described in the legend to Fig. 1. (C) Western blot analysis showing that wt and mt Upf1 proteins were expressed at similar levels. Cytoplasmic lysates prepared from NIH 3T3 B₂A₂ cells transfected with BBB^PTC in the absence (control) or presence of either wt Upf1 or mt Upf1 protein were resolved on a sodium dodecyl sulfate-8% polyacrylamide gel. The blot was probed with antibody against the Flag tag for detection of Upf1 and its mutant proteins and a control monoclonal antibody against α-tubulin for equal loading of protein samples.

Nonsense codon triggers accelerated deadenylation of β-globin mRNA in the cytoplasm, preceding the decay of the mRNA body. (A) Physical map of β-globin gene whose transcription is driven by the c-fos or Tet-regulated promoter. The open rectangles, thin lines, and thick lines depict exons, introns, and the 3′ flanking region of the rabbit β-globin gene, respectively. The sequences encoding the translation initiation codon (ATG) and the termination codon (TGA) are indicated. The arrow marks the point where the PTC was introduced. (B) Left, Northern blots showing deadenylation and decay of wild-type (BBB) and PTC-containing (BBB^PTC) β-globin mRNAs transcribed from the c-fos promoter in serum-induced NIH 3T3 cells. Middle, comparison of deadenylation rates of BBB and BBB^PTC mRNAs. Right, comparison of decay rates of BBB and BBB^PTC mRNAs. (C) Top, Northern blots showing deadenylation and decay of BBB and BBB^PTC mRNAs transiently transcribed from the Tet-regulated promoter following a transcription pulse in proliferating NIH 3T3 B₂A₂ cells. Bottom left, comparison of deadenylation rates of BBB and BBB^PTC mRNAs. Bottom right, comparison of decay rates of BBB and BBB^PTC mRNAs. (B and C) NIH 3T3 cells (B) or subline cells stably harboring tTA for the Tet-regulated promoter system (C) were transiently cotransfected with a control plasmid (pSVα/GAPDH) and one of the test plasmids (pBBB or pBBB^PTC). Total cytoplasmic mRNA was isolated at various time intervals after serum stimulation of quiescent cells (B) or after the addition of Tet (C) to proliferating cells and analyzed by Northern blot analysis (see Materials and Methods). α-globin/GAPDH control mRNAs were expressed constitutively and served as an internal standard. The times given above the blots correspond to hours after serum stimulation (B) or after Tet addition (C). Poly(A)⁻ RNA was prepared in vitro by treating RNA samples with oligo(dT) and RNase H. Poly(A) lengths were compared by calculating from the difference in electrophoretic mobility between each message and cognate poly(A)⁻ RNA. The quantitation of data was obtained by scanning the radioactive blots with an imager (Packard). Deadenylation and decay curves were plotted as described previously (34, 36). All curves shown in the plots are based on the average results of multiple experiments with standard deviations.

Targeted blockage of translation of PTC-containing β-globin mRNA impedes the deadenylation step and stabilizes the message. (A) Top, Northern blots showing deadenylation and decay of BBB^PTC and BBB^PTC+hp mRNAs. Bottom, comparison of deadenylation and decay rates of BBB^PTC and BBB^PTC+hp mRNAs. (B) Northern blots showing deadenylation and decay of BBB and BBB+hp mRNAs. Transient transfection, time course experiments, RNA isolation, poly(A)⁻ RNA preparation, poly(A) length comparisons, and data quantitation and plotting were as described in the legend to Fig. 1. (Note: deadenylation and decay curves for BBB^PTC mRNA are based on the results of multiple experiments, including the ones shown in this figure and in Fig. 1.)

Ectopic expression of UNR and NSAP1 blocks deadenylation of PTC-containing β-globin mRNA, leading to message stabilization. Shown are Northern blots demonstrating deadenylation and decay of BBB^PTC mRNAs in serum-stimulated NIH 3T3 cells in the absence (B, Control) or presence of ectopically expressed UNR (A, top left), NSAP1 (A, top right), or HuR (B, bottom) and deadenylation and decay of BBB mRNA in the presence of ectopically expressed UNR (A, bottom left) or NSAP1 (A, bottom right). Transient transfection, time course experiments, RNA isolation, and poly(A)⁻ RNA preparation were as described in the legend to Fig. 1.