Loss of CYP1A1 messenger RNA expression due to nonsense-mediated decay

Clones of the mouse hepatoma cell line Hepa1c1c7 (Hepa-1) with lesions in t he Cyp1a1 gene were isolated previously. A subset of these clones fails to express CYP1A1 mRNA even when treated with 2,3,7,8-tetrachlorodibenzo-p-dio xin, which induces this mRNA in wild-type Hepa-1 cells. The current investi gation sought an explanation for this phenotype in one of these clones, c33 . Loss of mRNA expression in c33 was shown to be caused by mutational chang es in the Cyp1a1 gene rather than by its epigenetic silencing. No mutations were identified in the 5' flanking region of the Cyp1a1 gene, containing t he promoter and dioxin-responsive enhancer sequences. A single nucleotide i nsertion occurred at nucleotide 418 in the coding region of one Cyp1a1 alle le, and a single nucleotide insertion occurred at nucleotide 465 in the oth er allele in c33. These sequence alterations were confirmed in the genomic DNA of the clone. Both insertions generate a premature termination codon at codon 172. This termination codon occurs in a position within the intron/e xon structure of the Cyp1a1 gene such that the encoded mRNA should be subje ct to "nonsense-mediated decay" (NMD). Inhibition of protein synthesis is k nown to reverse NMD. The protein synthesis inhibitors cycloheximide and pur omycin fully restored CYP1A1 mRNA expression to c33 cells, supporting the n otion that NMD degrades CYP1A1 mRNA in this strain. The mutations identifie d in the coding region of c33 provide an explanation, therefore, for its lo ss of both CYP1A1 enzymatic activity and inducible CYP1A1 mRNA expression.