Two mutations in the β-globin polyadenylylation signal reveal extended transcripts and new RNA polyadenylylation sites

Two mutations in the β-globin poly(A) signal were identified in Israeli patients with β⁺-thalassemia by sequence analysis following PCR. One is a point mutation (AATAAA → AATAAG) and the other is a 5-base-pair deletion (AATAAA → A - ). The mutant genes were used to investigate the function of the poly(A) signal in vivo and to evaluate the mechanism whereby these mutations lead to a thalassemic phenotype. Analysis of RNA derived from peripheral blood demonstrated the presence of elongated RNA species in patients carrying either mutation. Other aspects of RNA processing (initiation, splicing) were unimpaired. RNA obtained from the patients carrying the point mutation contained four discrete, extended RNA species, 1500-2900 nucleotides long, which were found to be polyadenylylated. Some normal cleavage-polyadenylylation was also observed. The 5-base-pair deletion completely abolished cleavage at the normal site. This deletion mutation resulted in a phenotype of β⁺-thalassemia, thus providing evidence that the extended mRNAs are translatable in vivo. Furthermore, additional transcripts, >5 kilobases, presumably mRNA precursors, were found in all RNA samples, including those of nonthalassemic controls. The extended transcripts of the poly(A) mutants, together with the high molecular weight precursors, suggest that the human β-globin gene transcription unit is significantly longer than previously recognized..