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.

Abstract

We have characterized a Mediterranean ,Bthalassemia allele containing a sequence change at codon 30 that alters both ,B-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G -. C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg -. 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 ofpre-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. and 3).The splicing of mRNA precursors requires at least two conserved sequences at the ends of introns: the 5' splice-site consensus sequence CAG/GTAAGT, which shows a striking complementarity to the 5'-terminal region ofU1 small nuclear RNA (snRNA), and the 3' splice-site consensus sequence Y"NCAG/ (4-6). Most of the naturally occurring mutations and in vitro-generated mutations that reduce the complementarity with U1 snRNA result in authentic 5' splice-site inactivation (for reviews, see refs. 7 and 8) and can activate cryptic sites (9-11). Biochemical data and complementation experiments also support the proposal that the 5' end of U1 snRNA interacts with the 5' splice site in mRNA precursors (12)(13)(14)(15). However, it is not possible to predict with confidence the effect of one mutation at a given 5' splice site. For instance, a number of point mutations in the 5' splice site of the large rabbit 3-globin intron do not severely inhibit splicing in transfected Hela cells or in vitro (10,16,17). These results could be interpreted by proposing that 5' splice sites whose sequences match best with the consensus can undergo point mutation without severe inactivation (18,19). Nevertheless, the strength of the 5' splice sites is probably not the only factor involved in the splice-site selection, which can be less dependent on match to the consensus than on the sequence context in which the 5' splice site resides (20). A possible explanation for these divergent observations could be that the individual contribution of exon or intron residues to the 5' splice site is not equivalent. However, the role of the nucleotides preceding the 5' splice site, which are moderately conserved, has not been extensively documented (8). It is striking that no natural mutations of the guanine at position -1 (present in 80% of cases for human genes) have been isolated.Here we describe the isolation and characterization of a G C mutation in the consensus sequence at the last nucleotide of exon 1...

Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis.

“…A splice junction mutation, which has been shown in other genes to result in abnormal RNA splicing (19)(20)(21)(22)(23), would be consistent with the decreased steady-state Gsa mRNA levels found in the affected members of kindred A (10). The coding frameshift mutation found in kindred B would prevent the generation of a normal full-length Gsa from the abnormal allele.…”

Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

“…As far as we know, there are only two examples of mutations in the consensus 5'-CAG-3' trinucleotide at the 3' terminal end of exons which inactivate the 5' splice donor site. Grandchamp et al reported that a G to A point mutation in the last position of exon 12 of the porphobilinogen deaminase gene leads to skipping of exon 12 in a case of acute intermittent porphyria (7), and one case of,-thalassemia has been reported to be due to a G-C transversion at the last position of exon 1 that drastically reduces splicing at the normal 5' splice site (21). We are not aware ofany other cases where mutations outside the splice site consensus sequences are known to induce exon skipping.…”

“…The amplification and detection methods were as described above. Trace amounts ofnormally spliced mRNA would be expected if normal splicing was not completely abolished (19,20,21). To detect this, the region of the agarose gel to which this would have migrated after electrophoresis was cut out.…”

Carbamyl phosphate synthetase I deficiency. One base substitution in an exon of the CPS I gene causes a 9-basepair deletion due to aberrant splicing.

“…The precedents are thalassemia, the spf ash mutation at the ornithine transcarbamylase locus of mouse, and ry5208 mutation at the rosy (xanthine dehydrogenase) locus of Drosophila melanogaster (15)(16)(17). ,B+ Thalassemia had the same G to C transversion at the last position of the exon.…”