Hu J, Connor J R
Department of Neuroscience and Anatomy, Penn State University College of Medicine, M. S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
J Neurochem. 1996 Aug;67(2):838-44. doi: 10.1046/j.1471-4159.1996.67020838.x.
Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that regulate expression of ferritin, erythroid 5-aminolevulinic acid synthase, and transferrin receptor through interaction with conserved RNA stem-loop structures called iron-responsive elements (IREs). Two IRPs (IRP1 and IRP2) have been reported. In the present study we provide evidence for and initial characterization of the IRPs in human brain. Two RNA-protein complexes were obtained by RNA band shift assay on cytoplasmic extracts from human brain. Competition studies indicate that the formations of the RNA-protein complexes are specific to the IRE structure. UV crosslinking of brain cytoplasmic extracts with ferritin IRE RNA transcripts revealed a single RNA-protein complex with a molecular mass of 110 kDa. A single band at 100 kDa was obtained with IRP1 antiserum on western blot analysis of brain cytoplasmic extracts, and a supershift in the RNA-protein complexes was observed with an IRP1 antiserum. Two cDNA clones were isolated from a human brain cDNA library with IRP1 cDNA probes, and both of these cDNA probes recognized a single mRNA species (4.0 kb) from human astrocytoma cells. Purified human brain IRP protein has a molecular mass of approximately 100 kDa and is capable of forming two RNA-protein complexes with ferritin IRE RNA and reacts strongly with IRP1 antiserum. These data indicate that IRP1 is predominant in the adult human brain and, in this tissue, is capable of forming a double IRE/IRP complex. This latter observation suggests the brain IRP undergoes posttranslational modification, the result of which may influence the stability of the IRE/IRP complex.
铁调节蛋白（IRPs）是一种细胞质RNA结合蛋白，通过与称为铁反应元件（IREs）的保守RNA茎环结构相互作用，调节铁蛋白、红细胞5-氨基酮戊酸合酶和转铁蛋白受体的表达。已报道了两种IRP（IRP1和IRP2）。在本研究中，我们提供了人类大脑中IRP的证据及初步特征。通过对人脑细胞质提取物进行RNA凝胶迁移试验，获得了两种RNA-蛋白质复合物。竞争研究表明，RNA-蛋白质复合物的形成对IRE结构具有特异性。人脑细胞质提取物与铁蛋白IRE RNA转录本的紫外线交联显示出一种分子量为110 kDa的单一RNA-蛋白质复合物。用人脑细胞质提取物进行蛋白质免疫印迹分析时，用IRP1抗血清获得了一条100 kDa的单带，并且观察到RNA-蛋白质复合物出现超迁移。用IRP1 cDNA探针从人脑cDNA文库中分离出两个cDNA克隆，这两个cDNA探针都识别来自人星形细胞瘤细胞的单一mRNA种类（4.0 kb）。纯化的人脑IRP蛋白分子量约为100 kDa，能够与铁蛋白IRE RNA形成两种RNA-蛋白质复合物，并与IRP1抗血清发生强烈反应。这些数据表明，IRP1在成人大脑中占主导地位，并且在该组织中能够形成双IRE/IRP复合物。后一观察结果表明，大脑IRP经历了翻译后修饰，其结果可能会影响IRE/IRP复合物的稳定性。
