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Intranuclear defect in beta-globin mRNA accumulation due to a premature
translation termination codon
K Takeshita, BG Forget, A Scarpa and EJ Benz
We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a
patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia.
Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's
erythroblasts suggested an intranuclear defect in both beta and Lepore
(delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA.
However, the nucleotide sequence of the beta O- 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 promotor boxes and the mRNA initiation (CAP) site, were
normal. The unexpected effect of this mutation on intranuclear beta-mRNA
synthesis in vivo was studied by insertion of the cloned gene into a
plasmid expression vector and transfection into tissue culture (COS-1)
cells. beta-Globin mRNA produced by the transfected cells was assessed by
S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to
tenfold less beta- mRNA than a normal beta-gene in both nuclear and
cytoplasmic RNA, simulating the results observed in vivo. Moreover, the
small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA
during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a
cause of the reduced accumulation. Cotransfection of the beta O-39
thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in
restoration of the beta O-39 mRNA accumulation to near-normal levels. On
the basis of these results, we suggest that the low levels of beta-mRNA
known to exist in the common form of beta O-thalassemia, beta O-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.
Volume 64,
Issue 1,
pp. 13-22,
07/01/1984
Copyright © 1984 by The American Society of Hematology
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