Intranuclear defect in β-globin mRNA accumulation due to a premature translation termination codon

Abstract

We have analyzed a cloned β0-thalassemia (β0-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and β0-thalassemia. Studies of 3H-uridine incorporation into β-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both β and Lepore (δβ) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the β0-thal gene revealed only a single base change in codon 39 (CAG → UAG), which created a premature translation termination codon. The 5' flanking sequence, including transcription promoter boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear β-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmic expression vector and transfection into tissue culture (COS-1) cells. β-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The β0-39 thalassemia gene generated five- to tenfold less β-mRNA than a normal β-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of β0-39 mRNA produced was as stable as normal β-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the β0-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the β0-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of β-mRNA known to exist in the common form of β0-thalassemia, β0-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.