β⁰-39 thalassemia gene: A premature termination codon causes β-mRNA deficiency without affecting cytoplasmic β-mRNA stability

The function of a β⁰-thalassemia globin gene with a premature termination codon at position 39 was studied in tissue culture cells using plasmic expression vectors. The thalassemic globin gene was isolated by molecular cloning from the bone marrow DNA of an Italian patient with severe thalassemia. Sequences upstream of the normal β-globin gene and the β⁰-39 globin gene were removed to 127 nucleotides (nt) 5' to the start site of transcription, thereby eliminating uncharacterized DNA sequences but preserving promoter function. To create a hybrid gene differing from the normal by only the single nt change in codon 39, the truncated 5' end of the β⁰-39 gene was recombined with the 3' end of the normal gene. The β⁰-39 substitution resulted in a 6-14-fold reduction in cytoplasmic RNA accumulation in transfected monkey kidney or HeLa cells. This decrease in mRNA did not appear to be due to instability, as the β-mRNA present 24-36 hours after transfection was stable for up to 24 hours in the presence of actinomycin D. In the presence of actinomycin D, the globin mRNA precursor disappeared, suggesting that globin gene transcription had been effectively blocked. We also examined a second globin mRNA with a premature termination codon; this RNA that arises from incorrect splicing of the transcript of a β⁺-24 thalassemia gene was as stable as the correctly spliced species. Thus, the presence of a premature termination codon does not necessarily after cytoplasmic mRNA stability, nor does cytoplasmic instability account for the quantitative deficiency of β-globin mRNA. Our observations suggest that a premature termination codon alters intranuclear stability and/or nuclear to cytoplasmic transport of the β-globin mRNA.