DMT1:Tissue specific expression & alternative splicing

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DESCRIPTION (provided by applicant): Insight into iron metabolism has grown rapidly in recent years with identification of proteins involved in iron absorption and trafficking, and recognition of the role of iron in gene expression. One recently identified protein is divalent metal ion transporter I (DMT1) which functions in iron absorption by the duodenal enterocyte and iron transport in the erythroblast. DMT1 mRNA is found in every tissue, but its role in tissues not involved in iron metabolism is unclear. Evaluation of DMT1 mRNA reveals alternative splicing at both the 5' and 3' ends of the molecule leading to at least 4 isoforms, which seem to be distributed differently in various tissues. In rodents, DMT1 gene mutation leads to hypochromic/microcytic anemia and iron deficiency, however, the only known human DMT1 mutation was found in a woman with hypochromic/microcytic anemia and iron overload. The human mutation predicts a conservative amino acid substitution, but its predominant effect is skipping of exon 12. In normal subjects, approximately 10% of reticulocyte DMT1 mRNA is exon 12 skipped transcript suggesting that the mutation exaggerates a "normal" process. Supratherapeutic concentrations of erythropoietin or hypoxia correct the phenotype of the patient's erythroid cells in vitro. How this correction occurs at the molecular level is unknown as are the specifics of why the human patient is iron overloaded. The details of how iron regulates DMT1 mRNA levels, and the roles that hypoxia, erythropoietin, and cytokines play are unclear. The function of exon 12 skipping, and its regulation also remain to be explored. This proposal addresses these issues by examining factors, which control DMT1 mRNA, and protein levels in various tissues. The effects of iron status, hypoxia, erythropoietin and cytokine levels on transcription, message stability and alternative splicing will be evaluated in human cell lines and primary erythroid cell cultures. Once these factors have been identified, the tissue specificity of exon 12 skipping, the association of exon 12 skipping with a specific transcript, and its modulation by factors which affect DMT1 mRNA levels will be examined. Appreciation of DMT1 regulation in various tissues will help further unravel its function(s) in tissues involved in iron metabolism, but also in tissues, which are not. This knowledge will be invaluable not only in treating anemic patients, but also in treating patients who are iron overloaded or in whom iron overload of specific tissues contributes to disease.