Figure 1.

Transient expression of R(844)C hUPF1 cDNA in COS cells abrogates the nonsense-mediated decrease in the abundance of β-globin mRNA. COS cells were transiently transfected with a pmCMV-Gl test plasmid (either Norm, which is nonsense-free, or 39Ter, which harbors a nonsense codon at amino acid position 39), the phCMV-MUP reference plasmid, and pCI-neo-hUPF1 [either Wt, which harbors an unmutagenized hUpf1p reading frame, or R(844)C, which harbors the arginine-to-cysteine change at amino acid position 844]. The amounts of each plasmid used were, respectively, 10 μg, 3 μg, and 37 μg, or 19 μg, 3 μg, and 28 μg. An appropriate amount of a fourth plasmid was added to each transfection to bring the total amount of introduced DNA to 50 μg. Nuclear and cytoplasmic RNA was purified (29, 30), and 40 μg were analyzed by blot hybridization to detect globin (Gl) RNA and mouse major urinary protein (MUP) RNA. Hybridization was quantitated by PhosphorImaging. The level of Gl mRNA from each mCMV-Gl allele was normalized to the level of MUP mRNA to provide a quantitative analysis. Normalized values then were calculated as a percentage of the normalized value of Gl Norm mRNA in the presence of either the Wt hUPF1 gene or the R844C hUPF1 gene or as a percentage of the normalized value of Gl 39Ter mRNA in the presence of the R844C hUPF1 gene, each of which was considered as 100. Percentages differed between two independently performed experiment by no more than 7%.