Abstract
The iron-responsive element binding protein (IRE-BP) is a cytosolic protein that binds a highly conserved sequence in the untranslated regions of mRNAs involved in iron metabolism including ferritin, transferrin receptor, and erythroid 5-aminolevulinate acid synthase. This conserved sequence is termed the iron-responsive element and is necessary for the post-transcriptional regulation of these mRNAs by iron. The rat liver IRE-BP was purified to homogeneity by chromatographic methods and partial amino acid sequence was obtained. A cDNA was isolated from a rat liver cDNA library and sequenced. The amino acid sequence deduced from the cDNA sequence corresponds to a protein of 889 amino acids with a predicted molecular weight of 97.946. The NH2-terminal sequence obtained by Edman degradation matched the deduced amino acid sequence obtained from the cDNA, confirming the translational start site. Rat liver IRE-BP shares 95% identity with human IRE-BP and 98% identity with mouse IRE-BP indicating that the IRE-BPs have remained highly conserved during evolution. The 5'-untranslated region is at least 236 nucleotides and contains interesting structural features including two direct repeats, an inverted repeat, and three small open reading frames. The rat IRE-BP mRNA is approximately 3600 nucleotides and is expressed in a variety of rat tissues including liver, spleen, and gut. Over the course of 16 h following an intraperitoneal injection of iron in rats. IRE-BP RNA binding activity decreases to 50% of control levels. The decrease in IRE-BP RNA binding activity in extracts from iron-treated rats is reversible by pretreatment of the extracts with reducing agents. The steady-state levels of IRE-BP mRNA remain constant during iron treatment. These data suggest that the decrease in IRE-BP RNA binding activity by iron in rat liver is due to post-translational changes in the RNA binding affinity of the IRE-BP and not due a decrease in the transcription of the IRE-BP gene or to the destabilization of the IRE-BP mRNA.
Highlights
From the $Program in HumanMolecular Biology and Genetics and the §Department of Medicine, University of Utah, Salt Lake City, Utah 84112
The IREBP is a RNA-binding proteinthat binds to a conserved RNA motif, the iron-responsive element (IRE), in either the 5’- or 3”untranslated regions of ferritin (18, 19), TfR (14, 20-22), and erythroid 5-aminolevulinate synthase mRNAs (23, 24). 5-Aminolevulinate synthase is the first enzyme in the heme biosynthetic pathway
Purification, Identification, andPartial Amino Acid Sequence of Rat Liver iron-responsive element binding protein (IRE-BP)-IRE-BP from rat liver was purified by chromatographic methods
Summary
Purification, Identification, andPartial Amino Acid Sequence of Rat Liver IRE-BP-IRE-BP from rat liver was purified by chromatographic methods (see "Materials and Methods"). The NH2- total rat liver RNA (Fig. 5A).A mRNA of about 3600 nt terminal sequence obtained by Edman sequencing matched hybridized to the IRE-BPcDNA and is similar to the size of the amino acid sequence predicted by the cDNA sequence, the human IRE-BP mRNA (35). Anoligonucleotide corresponding to thefirst 25 nt in the Regulation of the IRE-BP in Rat Liver-To determine if coding region produced two reverse transcription products of there is a correlationbetween changes in IRE-BP RNA bindabout 120 and 230 nt, indicating that the pSL-8cDNA clone ing activity and IRE-BP mRNA levels in the livers of rats was not full-length (data notshown). The 3”untranslated region of the IRE-BPcDNA contains 32P-labeledIRE, followed by resolution of the 1RE.IRE-BP
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