FIG. 1.

The nonsense-mediated reduction in TPI mRNA abundance is eliminated by deleting all six TPI introns and is restored by reinserting TPI intron 6. NIH 3T3 cells were transfected with the reference pmCMV-Gl construct and the pmCMV-TPI construct specified above each lane, where Ter indicates a nonsense codon and Δ specifies deletion of the designated introns. RNA was purified from total-cell (A), cytoplasmic (B), or nuclear (C) fractions with guanidine isothiocyanate and by cesium chloride centrifugation (12, 46), and 25 μg was analyzed by blot hybridization to DNA fragments ³²P labeled by random priming. TPI RNA was detected with a 299-bp NdeI-NcoI fragment that derives from the 3′ untranslated region of human TPI cDNA, and β-globin RNA was detected with a 170-bp BalI-DraI fragment that includes 158 bp of exon 3 from the mouse β^major-globin gene (11). Hybridization was quantitated with a PhosphorImager. The level of mRNA from each mCMV-TPI allele was normalized to the level of mCMV-Gl mRNA in order to control for variations in the efficiencies of cell transfection and RNA recovery. Normalized values for mCMV-TPI mRNAs that derived from constructs harboring a nonsense codon at position 189 and a deletion of either introns 1 through 6 or introns 1 through 5 [189Ter, Δ(introns 1–6), or 189Ter, Δ(introns 1–5), respectively] were then calculated as a percentage of the corresponding construct harboring a nonsense-free sequence [Norm, Δ(introns 1–6), or Norm, Δ(introns 1–5), respectively]. Values shown are an average of the values obtained from two independently performed transfections, which did not differ by more than 7%.