IRON REGULATION OF GENE EXPRESSION

铁对基因表达的调节

• 批准号: 3304571
• 负责人: Elizabeth Ann Leibold
• 金额: $ 13.18万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304571
• 关键词: binding proteins; complementary DNA; ferritin; gene expression; genetic library; genetic regulation; genetic transcription; iron metabolism; laboratory rabbit; laboratory rat; liver cells; liver metabolism; messenger RNA; molecular cloning; neoplastic cell culture for noncancer research; protein biosynthesis; protein sequence; transferrin receptor

Iron is an essential element and is required by most cells for survival and growth. In excess, iron is toxic to cells. Consequently, its level in cells is tightly regulated. Mammalian cells maintain stable cytosolic concentrations of free iron both by regulating their uptake of iron via the transferrin receptor (TfR) and by sequestering intracellular iron into ferritin. Ferritin synthesis is induced by iron through a mechanism which shifts latent ferritin mRNA to actively translating polysomes. TfR synthesis is decreased by iron, which promotes destabilization of TfR mRNA. The coordinate regulation of ferritin and TfR synthesis by iron is controlled by iron responsive elements, or IREs. These IREs are located in the 5'- and 3'-untranslated regions of ferritin and TfR mRNAs, respectively. The IREs form a characteristic stem-loop structure and bind cytosolic proteins, the iron responsive proteins or IRE-BPs. Two such proteins, IRE-BP Bl and IRE-BP B2, have been characterized in part. The proposed experiments are aimed at determining the mechanisms through which ferritin and TfR mRNAs are coordinately regulated by the IRE-BPs. The first specific aim is to isolate an IRE-BP cDNA by screening a rat liver lambda cDNA library with oligonucleotides derived from amino acid sequences of IRE-BP, the principal rat IRE-BP. The cDNA sequence will be determined and the deduced amino acid sequence will be compared to amino acid sequences of other proteins to identify conserved regions of the IRE-BP that may be functionally important in RNA-protein recognition. The second specific aim is to study the expression of IRE-BP B1 in rat hepatoma cells. The IRE-BP cDNA will be used to determine if IRE-BP B1 expression responds to changes in intracellular iron and/or heme levels and to determine if transcriptional or post-transcriptional mechanisms are involved in the regulation of IRE-BP B1 synthesis. The third specific aim is test the predicted secondary of the ferritin and TfR IREs by synthesizing IRE RNAs containing altered nucleotide sequences and testing these mutants for IRE-BP B1 binding. These studies will permit the correlation of IRE structure with biological function. The fourth specific aim is to determine the function of a second protein in rat liver, IRE-BP B2. IRE-BP B2 protein from rat liver will be isolated and sequenced, and its function and the functional determinants in its structure will be characterized. The fifth and last specific aim is to determine if there are other factors involved in the regulation of TfR mRNA by iron. A cell-free system will be developed to identify polysomal or cytosolic factors other than IRE-BPS, that may specifically affect the degradation of TfR mRNA.

铁是必不可少的元素，大多数细胞都需要生存和 生长。过量的铁对细胞有毒。因此，其在细胞中的水平 受到严格调节。哺乳动物细胞保持稳定的胞质 游离铁的浓度都通过调节其通过 转铁蛋白受体（TFR）和通过隔离细胞内铁 铁蛋白。铁合成是由铁通过一种机制诱导的 将潜在的铁蛋白mRNA转移到积极翻译多聚体。 TFR 铁通过铁减少，从而促进TFR mRNA的不稳定。 铁IS的铁蛋白和TFR合成的坐标调节 由铁响应元素或IRES控制。这些IRES位于 铁蛋白和TFR mRNA的5'-和3'-非翻译区域， 分别。 IRES形成特征性的茎环结构并结合 胞质蛋白，铁反应蛋白或IRE-BPS。两个这样的 蛋白质IRE-BP BL和IRE-BP B2已被部分表征。这 提出的实验旨在确定该机制 铁蛋白和TFR mRNA由IRE-BPS协调调节。 第一个具体目的是通过筛选大鼠隔离IRE-BP cDNA 肝lambda cDNA文库，衍生自氨基酸的寡核苷酸 IRE-BP的序列，主要大鼠IRE-BP。 cDNA序列将是 确定并将推导的氨基酸序列与氨基进行比较 其他蛋白质的酸序列，以鉴定 IRE-BP在RNA蛋白识别中可能在功能上很重要。这 第二个具体目的是研究大鼠肝癌中IRE-BP B1的表达 细胞。 IRE-BP cDNA将用于确定IRE-BP B1是否表达 响应细胞内铁和/或血红素水平的变化以及 确定转录或转录后机制是 参与IRE-BP B1合成的调节。第三个特定目标 测试由铁蛋白和TFR IRES的预测次级测试 合成含有改变核苷酸序列和测试的IRE RNA 这些用于IRE-BP B1结合的突变体。这些研究将允许 IRE结构与生物功能的相关性。第四个特定 目的是确定第二蛋白在大鼠肝脏中的功能 B2。来自大鼠肝脏的IRE-BP B2蛋白将被分离和测序，并且 它的功能及其结构中的功能决定因素将是 特征。第五和最后一个具体目的是确定是否存在 铁涉及的其他因素通过铁调节TFR mRNA。无细胞 将开发系统以识别其他多个或胞质因子其他 比IRE-BPS，可能特别影响TFR mRNA的降解。