IRON REGULATION OF GENE EXPRESSION

铁是一种必需元素，是大多数细胞生存所必需的， 增长过量的铁对细胞是有毒的。因此，其在细胞中的水平 受到严格管制。哺乳动物细胞维持稳定的胞浆 游离铁的浓度都通过调节其铁的吸收，通过 转铁蛋白受体（TfR）和螯合细胞内铁进入 铁蛋白铁蛋白的合成是由铁通过一种机制诱导的， 将潜伏的铁蛋白mRNA转移到主动翻译的多核糖体。TFR 铁降低了TfR mRNA的合成，铁促进了TfR mRNA的不稳定。 铁对铁蛋白和TfR合成的协调调节是 由铁反应元件或IRES控制。这些IRE位于 铁蛋白和TfR mRNA的5 '-和3'-非翻译区， 分别IRE形成特征性茎环结构并结合 细胞溶质蛋白、铁响应蛋白或IRE-BP。两个这样 蛋白质IRE-BP B1和IRE-BP B2已被部分表征。的 拟议的实验旨在确定通过哪些机制， 铁蛋白和TfR mRNA由IRE-BP协同调节。 第一个具体目的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λ 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。 其他蛋白质的氨基酸序列，以鉴定 IRE-BP可能在RNA-蛋白质识别中具有重要功能。的 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 响应于细胞内铁和/或血红素水平的变化， 确定转录或转录后机制是否 参与调节IRE-BP B1的合成。第三个具体目标 通过以下方法检测预测的铁蛋白和TfR IRE的次级 合成含有改变的核苷酸序列的IRE RNA并测试 这些突变体的IRE-BP B1结合。这些研究将使 IRE结构与生物学功能的相关性。第四特定 目的是确定大鼠肝脏中第二种蛋白质IRE-BP的功能 B2.将从大鼠肝脏分离IRE-BP B2蛋白并测序， 其功能及其结构中的功能决定因素将是 表征了第五个也是最后一个具体目标是确定是否有 铁对TfR mRNA的调控中涉及的其他因素。无细胞 将开发系统来鉴定多聚体或胞质因子， 比IRE-BPS更能特异性地影响TfR mRNA的降解。

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.