Nonsense mutations in the human beta-globin gene lead to unexpected levelsof cytoplasmic mRNA accumulation

Generally nonsense codons 50 bp or more upstream of the 3'-most intron of t he human beta -globin gene reduce mRNA abundance. In contrast, dominantly i nherited beta -thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were st ably transfected with human beta -globin genes mutated within each of the 3 exons, namely at codons IS(TGG-->TGA), 39 (C-->T), or 127 (C-->T). Primer extension analysis after erythroid differentiation induction showed codon 1 27 (C-->T) mRNA accumulated in the cytoplasm at approximately 20% the norma l mRNA level. Codon 39 (C-->T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG-->TGA) mRNA accumulated at approx imately 90%, Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied, High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsen se mutation and the genomic background in which this mutation occurs. To in vestigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cl oned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high le vels, whereas the mutation at codon 26 led to low mRNA levels. These findin gs suggest that nonsense-mediated mRNA decay is not exclusively dependent o n the localization of mutations relative to the 3'-most intron, Other facto rs may also contribute to determine the cytoplasmic nonsense-mutated mRNA l evel in erythroid cells. (C) 2000 by The American Society of Hematology.