Evolutionarily Conserved Alternative Pre-mRNA Splicing Regulates Structure and Function of the Spectrin-Actin Binding Domain of Erythroid Protein 4.1

• Published in: Blood Vol. 86; no. 11; pp. 4315 - 4322
• Main Authors: Winardi, Ricky, Discher, Dennis, Kelley, Clare, Zon, Len, Mays, Karen, Mohandas, Narla, Conboy, John G.
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01-12-1995; The Americain Society of Hematology
• Subjects: Actins - metabolism; Alternative Splicing - genetics; Amino Acid Sequence; Animals; Base Sequence; Binding Sites - genetics; Biological and medical sciences; Biological Evolution; Blood coagulation. Blood cells; Conserved Sequence; Cytoskeletal Proteins; DNA, Complementary - genetics; Erythrocyte Membrane - metabolism; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Humans; In Situ Hybridization; Mammals; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular and cellular biology; Molecular Sequence Data; Neuropeptides; Phylogeny; RNA Precursors - genetics; RNA Precursors - metabolism; Spectrin - metabolism; Xenopus laevis; Spectrin; Alternative splicing; Membrane protein; Erythroid cell; Molecular structure; Frog; Amphibia; Contractile protein; Actins; Glycoproteins; Salientia; Binding site; Phylogeny; Biological activity; Vertebrata; Regulation(control); Exon; Animal
• DOI: 10.1182/blood.V86.11.4315.bloodjournal86114315
• EISSN: 1528-0020
• ISSN: 0006-4971

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21–amino acid peptide required for high-affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Here we show that key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes showed that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrated specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison showed two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos showed high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding.
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