Abnormal RNA splicing causes one form of α thalassemia

Abstract The α2-globin gene from α patient with a thalassemia contains a pentanucleotide deletion in intron 1 immediately adjacent to exon 1. We tested the functional consequences of this mutation by introducing the thalassemic gene, along with its normal counterpart as a control, into cultured monkey cells on SV40 plasmid vectors. Both genes are expressed, at similar levels, into globin RNA with the correct 5′ and 3′ ends. However, while most of the normal transcripts are appropriately processed, the thalassemic transcripts are abnormally spliced from α 5′ donor site in the middle of exon 1 to the normal 3′ acceptor site. This results in the synthesis of a truncated RNA incapable of encoding a normal globin polypeptide. The alternative donor, also used at a low level in monkey cells transfected with the normal gene, shows strong homology to the consensus donor sequence characteristic of many eucaryotic splice junctions. No unspliced or partially spliced thalassemic RNA was detected, indicating that recognition of this site is efficient and does not block removal of intron 2. The alternatively spliced RNA was also found in bone marrow RNA from the α-thalassemic patient, although not in that from a normal individual or a β + -thalassemic patient. The thalassemic phenotype of the patient therefore results from abnormal RNA splicing owing to the deletion of the first splice donor signal.

[1]  P. Leder,et al.  Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Orkin,et al.  Linkage of β-thalassaemia mutations and β-globin gene polymorphisms with DNA polymorphisms in human β-globin gene cluster , 1982, Nature.

[3]  P. A. Biro,et al.  Base substitution in an intervening sequence of a beta+-thalassemic human globin gene. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[4]  P. Thomas,et al.  Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Sharp,et al.  Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids , 1977, Cell.

[6]  B. Lewin.  Alternatives for splicing: Recognizing the ends of introns , 1980, Cell.

[7]  T. Maniatis,et al.  Identification of DNA sequences required for transcription of the human α1-globin gene in a new SV40 host-vector system , 1981, Cell.

[8]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[9]  S. Orkin,et al.  Nonsense and frameshift mutations in beta 0-thalassemia detected in cloned beta-globin genes. , 1981, The Journal of biological chemistry.

[10]  S. Orkin,et al.  The duplicated human α-globin genes: Their relative expression as measured by RNA analysis , 1981, Cell.

[11]  Judith A. Kantor,et al.  Beta thalassemia: Mutations which affect processing of the β-globin mRNA precursor , 1980, Cell.

[12]  J. T. Wilson,et al.  Nucleotide sequence of the coding portion of human alpha globin messenger RNA. , 1980, The Journal of biological chemistry.

[13]  Y W Kan,et al.  beta 0 thalassemia, a nonsense mutation in man. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Orkin,et al.  Mutation in an intervening sequence splice junction in man. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Maniatis,et al.  The chromosomal arrangement of human α-like globin genes: Sequence homology and α-globin gene deletions , 1980, Cell.

[16]  R. Flavell,et al.  β + Thalassemia: Aberrant splicing results from a single point mutation in an intron , 1981, Cell.

[17]  J. Darnell.  Implications of RNA-RNA splicing in evolution of eukaryotic cells. , 1978, Science.

[18]  W. Ford Doolittle,et al.  Genes in pieces: were they ever together? , 1978, Nature.

[19]  T. Maniatis,et al.  Amplification and characterization of a β-globin gene synthesized in vitro , 1976, Cell.

[20]  D. J. Weatherall,et al.  Recent developments in the molecular genetics of human hemoglobin , 1979, Cell.

[21]  P. Sharp.  Speculations on RNA splicing , 1981, Cell.

[22]  P. Leder,et al.  A mouse globin gene promoter is functional in SV40 , 1980, Cell.

[23]  D. Kemp,et al.  Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. F. Weaver,et al.  Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. , 1979, Nucleic acids research.

[25]  D. Hamer,et al.  Regulation in vivo of a cloned mammalian gene: cadmium induces the transcription of a mouse metallothionein gene in SV40 vectors. , 1982, Journal of molecular and applied genetics.

[26]  J. Banerji,et al.  Expression of a β-globin gene is enhanced by remote SV40 DNA sequences , 1981, Cell.

[27]  P. Leder,et al.  SV40 recombinants carrying rabbit β-globin gene coding sequences , 1979, Cell.

[28]  C Benoist,et al.  Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[29]  W. Gilbert.  Why genes in pieces? , 1978, Nature.

[30]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[31]  S. Weissman,et al.  Abnormally spliced messenger RNA in erythroid cells from patients with β + thalassemia and monkey cells expressing a cloned β +-thalassemic gene , 1982, Cell.

[32]  H. Lazarus,et al.  The molecular basis of α-thalassemias: Frequent occurrence of dysfunctional α loci among non-Asians with Hb H disease , 1979, Cell.