Kohler S A, Menotti E, Kühn L C
Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges s/Lausanne, Switzerland.
J Biol Chem. 1999 Jan 22;274(4):2401-7. doi: 10.1074/jbc.274.4.2401.
Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells.
铁调节蛋白（IRPs）通过与铁反应元件（IREs）结合来控制铁代谢中几种蛋白质的合成，IREs是相应mRNA非翻译区（UTR）中的一种发夹结构。IRPs与5'UTR中的IREs结合会抑制铁蛋白重链和轻链、红细胞δ-氨基乙酰丙酸合酶、线粒体乌头酸酶以及果蝇琥珀酸脱氢酶b的翻译，而IRP与转铁蛋白受体mRNA的3'UTR中的IREs结合会延长mRNA的半衰期。为了鉴定IRPs的新靶点，我们设计了一种方法，利用重组IRP-1作为亲和基质来富集含IRE的mRNA。通过RNA-蛋白质凝胶迁移试验筛选从富集mRNA建立的cDNA文库。这在一种肝脏特异性小鼠mRNA的3'UTR中揭示了一个新的IRE样序列。新鉴定的cDNA编码一种与植物乙醇酸氧化酶（GOX）具有高度同源性的蛋白质。在细菌中表达的重组蛋白表现出GOX酶活性。因此，该cDNA代表首个脊椎动物GOX同源物。小鼠GOX中的IRE样序列在室温下与IRPs表现出强结合。然而，它与功能性IREs的区别在于其上部茎的中间存在错配，并且在细胞中不赋予铁依赖性调节。
