Alphonse L. Scarpa

Structure and Organization of the Human Ankyrin-1 Gene BASIS FOR COMPLEXITY OF PRE-mRNA PROCESSING

Ankyrin-1 (ANK-1) is an erythrocyte membrane protein that is defective in many patients with hereditary spherocytosis, a common hemolytic anemia. In the red cell, ankyrin-1 provides the primary linkage between the membrane skeleton and the plasma membrane. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hereditary spherocytosis patients, we cloned the human ANK-1 chromosomal gene. Characterization of theANK-1 gene genomic structure revealed that the erythroid transcript is composed of 42 exons distributed over ∼160 kilobase pairs of DNA. Comparison of the genomic structure with the protein domains reveals a near-absolute correlation between the tandem repeats encoding the membrane-binding domain of ankyrin with the location of the intron/exon boundaries in the corresponding part of the gene. Erythroid stage-specific, complex patterns of alternative splicing were identified in the region encoding the regulatory domain of ankyrin-1. Novel brain-specific transcripts were also identified in this region, as well as in the “hinge” region between the membrane-binding and spectrin-binding domains. Utilization of alternative polyadenylation signals was found to be the basis for the previously described, stage-specific 9.0- and 7.2-kilobase pair transcripts of theANK-1 gene.

A nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Metabolism of Structurally Abnormal mRNAs Resulting from β-Thalassemia Mutationsa

The thalassemia syndromes arise from alterations in or near the globin genes that either delete the genes or cause impairment of transcription, processing, or translation of globin mRNAs.’, The impressive diversity of mutations capable of producing thalassemia is apparent from other manuscripts in this volume. The most common forms of /3-thalassemia, in terms of the number of patients affected, are those in which mutations alter processing of mRNA precursors or translation of mature &globin mRNA.3s In these cases, most or all of the globin mRNA generated by the defective globin gene is incapable of translation because of the presence of “in-phase’’ premature translation termination codons. FIGURES 1 and 2 show two examples of common forms of thalassemia in which most or all of the final mRNA products are, nontranslatable. FIGURE 1 illustrates a mutation causing alternative splicing of the P-mRNA prec~rsors.’.~ The abnormal pathway is used for the processing of 90% of the premRNA transcripts; the abnormally processed mRNA contains an in-phase terminator codon, as shown in the diagram. FIGURE 2 shows a single base substitution that directly alters a glutamic acid codon in position 39 to a premature translation termination codon. In this manuscript, we review some of our recent studies concerning the metabolism of these nontranslatable mRNAs. In forms of thalassemia characterized by the generation of premature translation termination codons, the amount of /3-mRNA is greatly reduced even though the

789 ABNORMAL METABOLISM OF β mRNA IN β+-THALASSEMIA (β-THAL)

We have studied mechanisms causing reduced g mRNA accumulation in β-thal. One patient was studied by recombinant DNA cloning, nucleotide sequencing and in vitro functional analysis of the β-thal gene, and five others, by analysis of newly synthesized mRNA after pulse-chase labeling of erythroblasts with 3H-uridine. The cloned β-thal gene exhibited normal transcription in a cell-free system. The only nucleotide sequence abnormality occurs within the small intervening sequence (intron) and creates a potential anomalous splicing site in the mRNA precursor, suggesting a structural basis for defective processing. In four additional patients, defective β mRNA processing was also observed. The initial β/α 3H mRNA ratios of pulse-labeled RNA were normal, indicating normal transcription, but abnormally high accumulation of unprocessed β mRNA precursur sequences (introns) occurred in each case. A fifth patient exhibited normal 3H β mRNA synthesis and processing in nuclear RNA, but β mRNA in cytoplasm declined to steady-state levels during a 20-hour “chase,” indicating cytoplasmic instability. These studies identify at least two distinct post-transcriptional lesions in β mRNA metabolism in β-thal: inefficient processing of introns and cytoplasmic instability of mature β mRNA.