Abstract
Linkage between transmembrane proteins and the spectrin-based cytoskeleton is necessary for membrane elasticity of red blood cells. Mutations of the proteins that mediate this linkage result in various types of hemolytic anemia. Here we report a novel N-ethyl-N-nitrosourea−induced mutation of ankyrin-1, named hema6, which causes hereditary spherocytosis in mice through a mild reduction of protein expression. The causal mutation was traced to a single nucleotide transition located deep into intron 13 of gene Ank1. In vitro minigene splicing assay revealed two abnormally spliced transcripts containing cryptic exons from fragments of Ank1 intron 13. The inclusion of cryptic exons introduced a premature termination codon, which leads to nonsense-mediated decay of the mutant transcripts in vivo. Hence, in homozygous mice, only wild-type ankyrin-1 is expressed, albeit at 70% of the level in wild-type mice. Heterozygotes display a similar hereditary spherocytosis phenotype stemming from intermediate protein expression level, indicating the haploinsufficiency of the mutation. Weakened linkage between integral transmembrane protein, band 3, and underlying cytoskeleton was observed in mutant mice as the result of reduced high-affinity binding sites provided by ankyrin-1. Hema6 is the only known mouse mutant of Ank1 allelic series that expresses full-length canonical ankyrin-1 at a reduced level, a fact that makes it particularly useful to study the functional impact of ankyrin-1 quantitative deficiency.
Highlights
Linkage between transmembrane proteins and the spectrin-based cytoskeleton is necessary for membrane elasticity of red blood cells
Hema6, an ENU-induced mutant phenotype of hereditary spherocytosis with mild hemolysis In an effort to identify proteins with nonredundant function in erythropoiesis, we established a genetic screen for abnormal red cell production and survival in mutant mice generated by large-scale ENU mutagenesis (Arnold et al 2012; Krieg et al 2011)
The index hema6 mouse was detected in the screen by its reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin but increased counts of red blood cell (RBC)
Summary
Linkage between transmembrane proteins and the spectrin-based cytoskeleton is necessary for membrane elasticity of red blood cells. RBC2 red blood cells exhibited total disruption of membrane skeleton architecture, and the phenotype was manifested as a recessive trait of severe hemolytic HS in mice (Rank et al 2009) Both Ank1E924X and Ank1MRI23420 lines are hypomorphic mutations, resulting in truncated ankyrin-1 proteins lacking both spectrin-binding domain and C-terminal regulatory domain, whereas band 3-binding domain remains intact in Ank1E924X (Hughes et al 2011) or partially affected in Ank1MRI23420 (Greth et al 2012). We report the identification and characterization of a novel ENU-induced mutation in Ank named hema6 Both heterozygous and homozygous animals exhibit characteristic features of hereditary spherocytosis, such as increased RBC counts, low RBC mean corpuscular volume (MCV), increased osmotic fragility, and shortened life span of RBCs in vivo. The analysis of these mice highlights the importance of optimal ankyrin-1 protein quantity in maintaining erythrocyte membrane stability
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