DMT1:Tissue specific expression & alternative splicing

DMT1：组织特异性表达

• 批准号: 7031607
• 负责人: Martha P. Mims
• 金额: $ 14.65万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031607
• 关键词: RNA splicing; biological transport; cell line; clinical research; cytokine; erythropoietin; gene expression profiling; human tissue; hypoxia; iron metabolism; iron poisoning; messenger RNA; transcription factor; transport proteins

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.

描述(申请人提供)：近年来，随着铁吸收和运输所涉及的蛋白质的确定，以及铁在基因表达中的作用的认识，对铁代谢的洞察力迅速增长。最近发现的一种蛋白质是二价金属离子转运蛋白I(DMT1)，它在十二指肠肠上皮细胞对铁的吸收和红细胞中铁的运输中发挥作用。DMT1在每个组织中都有表达，但它在不参与铁代谢的组织中的作用尚不清楚。对DMT1mRNA的评估显示，在分子的5‘和3’端都有选择性剪接，导致至少4种异构体，它们似乎在不同的组织中分布不同。在啮齿动物中，DMT1基因突变会导致低色素性/小细胞性贫血和铁缺乏，然而，唯一已知的人类DMT1突变是在一名患有低色素性/小细胞性贫血和铁负荷过高的女性身上发现的。人类的突变预示着保守的氨基酸替代，但它的主要作用是跳过外显子12。在正常人中，大约10%的网织红细胞DMT1 mRNA是外显子12跳过的转录本，这表明该突变夸大了“正常”的过程。超治疗浓度的促红细胞生成素或低氧在体外纠正患者红系细胞的表型。这种纠正是如何在分子水平上发生的尚不清楚，也不清楚为什么人类患者铁超载的具体原因。铁如何调节DMT1mRNA水平的细节，以及缺氧、促红细胞生成素和细胞因子所起的作用尚不清楚。12号外显子跳跃的功能及其调控也有待进一步研究。这项建议通过检查控制DMT1mRNA的因素和不同组织中的蛋白质水平来解决这些问题。铁状态、低氧、促红细胞生成素和细胞因子水平对转录、信息稳定性和选择性剪接的影响将在人类细胞系和原代红系细胞培养中进行评估。一旦确定了这些因素，将研究外显子12跳过的组织特异性，外显子12跳过与特定转录本的关联，以及影响DMT1mRNA水平的因素对其的调控。了解DMT1在各种组织中的调节作用将有助于进一步揭示其在参与铁代谢的组织中的功能(S)，也有助于进一步揭示其在非铁代谢组织中的作用。这一知识不仅在治疗贫血患者方面将是无价的，而且在治疗铁超载或特定组织铁超载导致疾病的患者方面也是如此。