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.

铁是一种必需元素，大多数细胞都需要铁来维持生存 生长。过量的铁对细胞有毒。因此，其在细胞中的水平 受到严格监管。哺乳动物细胞保持稳定的胞质 通过调节铁的吸收来调节游离铁的浓度 转铁蛋白受体 (TfR) 并通过将细胞内铁隔离到 铁蛋白。铁蛋白的合成是由铁通过以下机制诱导的： 将潜在的铁蛋白 mRNA 转变为主动翻译的多核糖体。转铁蛋白受体 铁会减少合成，从而促进 TfR mRNA 的不稳定。 铁对铁蛋白和TfR合成的协调调节是 由铁反应元件（IRE）控制。这些 IRE 位于 铁蛋白和 TfR mRNA 的 5'-和 3'-非翻译区， 分别。 IRE 形成特有的茎环结构并结合 胞质蛋白、铁反应蛋白或 IRE-BP。两个这样的 蛋白质IRE-BP B1和IRE-BP B2已部分表征。这 拟议的实验旨在确定机制 铁蛋白和 TfR mRNA 受 IRE-BP 协调调节。 第一个具体目标是通过筛选大鼠来分离 IRE-BP cDNA 含有源自氨基酸的寡核苷酸的肝脏 lambda cDNA 文库 IRE-BP 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酸序列进行比较 其他蛋白质的酸序列以识别保守区域 IRE-BP 在 RNA 蛋白质识别中可能具有重要的功能。这 第二个具体目的是研究IRE-BP B1在大鼠肝癌中的表达 细胞。 IRE-BP cDNA 将用于确定 IRE-BP B1 表达是否 对细胞内铁和/或血红素水平的变化做出反应 确定转录或转录后机制是否 参与 IRE-BP B1 合成的调节。第三个具体目标 测试预测的铁蛋白和 TfR IRE 的次级 合成含有改变的核苷酸序列的 IRE RNA 并进行测试 这些突变体用于 IRE-BP B1 结合。这些研究将允许 IRE结构与生物功能的相关性。第四具体 目的是确定大鼠肝脏中第二种蛋白质 IRE-BP 的功能 B2.来自大鼠肝脏的 IRE-BP B2 蛋白将被分离并测序，并且 它的功能和结构中的功能决定因素将是 特点。第五个也是最后一个具体目标是确定是否存在 其他参与铁调节 TfR mRNA 的因素。无细胞 将开发系统来识别多聚体或胞质因子 与 IRE-BPS 相比，这可能特别影响 TfR mRNA 的降解。