Henderson B R, Seiser C, Kühn L C
Swiss Institute for Experimental Cancer Research, Epalinges s/Lausanne.
J Biol Chem. 1993 Dec 25;268(36):27327-34.
Iron regulatory factor (IRF) is a cytoplasmic RNA-binding protein involved in regulating iron homeostasis. IRF controls expression of ferritin and transferrin receptor post-transcriptionally via specific binding to stem-loop iron-responsive elements (IREs) located in the untranslated regions of the respective mRNAs. We have confirmed by RNA band-shift analysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulated by intracellular iron levels. This faster migrating complex appears to represent a specific interaction between the ferritin IRE and an iron-regulated protein that is distinct from IRF, as concluded from the following lines of evidence. First, UV cross-linking and partial digestion with different proteases revealed different peptide patterns for the two IRE-protein complexes. Second, antiserum raised against IRF peptides immunoprecipitated only authentic IRF and not the protein of the faster migrating complex, as determined by band-shift analysis. Following separation of the two IRE-binding proteins by ion-exchange chromatography, only the IRF-containing fraction reacted with the antibodies on Western blots. The second protein binds IREs with an affinity similar to that of IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. UV cross-linking experiments indicate that this second protein, tentatively named IRFB, has a molecular mass of approximately 105 kDa. Analysis of mouse tissues revealed differences in the distribution of IRF and IRFB. Whereas IRF protein and IRE binding activity were predominant in liver, intestine, and kidney, the IRFB protein(s) revealed highest binding activity in intestine and brain. Our data support the existence of two distinct iron-regulated IRE-binding proteins in rodents.
铁调节因子（IRF）是一种细胞质RNA结合蛋白，参与调节铁稳态。IRF通过与位于各自mRNA非翻译区的茎环铁反应元件（IRE）特异性结合，在转录后控制铁蛋白和转铁蛋白受体的表达。我们通过RNA凝胶迁移分析证实，在不同啮齿动物细胞提取物中观察到的第二种IRE-蛋白复合物，与IRF一样，受细胞内铁水平的调节。这种迁移速度更快的复合物似乎代表了铁蛋白IRE与一种不同于IRF的铁调节蛋白之间的特异性相互作用，以下证据支持这一结论。首先，紫外线交联和用不同蛋白酶进行部分消化显示，两种IRE-蛋白复合物的肽谱不同。其次，通过凝胶迁移分析确定，针对IRF肽产生的抗血清仅免疫沉淀了真实的IRF，而没有沉淀迁移速度更快的复合物中的蛋白质。通过离子交换色谱分离两种IRE结合蛋白后，只有含IRF的部分在Western印迹上与抗体发生反应。如与铁蛋白IRE和相关茎环RNA的竞争所示，第二种蛋白与IRE的结合亲和力与IRF相似。紫外线交联实验表明，这种第二种蛋白暂命名为IRFB，分子量约为105 kDa。对小鼠组织的分析揭示了IRF和IRFB分布的差异。虽然IRF蛋白和IRE结合活性在肝脏、肠道和肾脏中占主导地位，但IRFB蛋白在肠道和大脑中显示出最高的结合活性。我们的数据支持在啮齿动物中存在两种不同的铁调节IRE结合蛋白。
