Molecular cloning of mouse glycolate oxidase. High evolutionary conservation and presence of an iron-responsive element-like sequence in the mRNA.
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.
Kohler
S A
SA
Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges s/Lausanne, Switzerland.
Menotti
E
E
Kühn
L C
LC
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D019143:Evolution, Molecular; D006801:Humans; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase; D035941:Iron Regulatory Protein 1