A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.

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Abstract
We have characterized a Mediterranean .beta.-thalassemia allele containing a sequence change at codon 30 that alters both .beta.-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G .fwdarw. C transversion at position-1 of intron 1 reduces severely the utilization of the normal 5'' splice site since thelevel of the Arg .fwdarw. Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position-1 of the CAG/GTAAGT consensus sequence had been isolated previously, we investigated the role of this nucleotide in the constitution of an active 5'' splice site by studying the splicing of the pre-mRNA in cell-free extracts. We demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Our results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.