ABSTRACT
Ferritin expression is regulated at the level of translation (reviewed in [1]). Ferritin genes contain motifs known as iron-responsive elements (IREs) that assume a stemloop structure and in the 5′ untranslated region (5′UTR) between the 5′ cap site, where assembly of the translation apparatus begins, and the beginning of the coding region, where the translation complex is fully assembled. Thus, the partially assembled translation apparatus must scan through the IRE sequence before protein biosynthesis can begin (2). When the cell is iron-replete, ferritin synthesis proceeds unimpeded, and excess iron is stored within the spherical ferritin molecule. However, when the cell is depleted of iron, ferritin synthesis is markedly decreased. The stem-loop is the binding site for the cytosolic iron-sensing protein, the IRE-BP. Binding of this protein can be detected in in vitro assays in which the IRE is radiolabeled and mixed with cytosolic extracts containing the IRE-BP. When the mixture of the cytosolic extract and radiolabeled probe is resolved on a nondenaturing gel (3), the protein binds and shifts the radiolabeled RNA to a new position in the gel. The assay is quantitative, and more IRE-BP binds to the IRE per unit of lysate when the cells used to make cytosolic extract are iron-deprived prior to lysis (5). The total amount of IRE-BP does not appear to change, since binding activity can be induced in lysates by the addition of high amounts of reducing reagents (5). Furthermore, Western blotting and quantitative immunoprecipitations show that the total amount of protein has not significantly changed (6). These results contradict results that have been obtained by others (7), in which binding of heme to the IRE-BP is thought to induce degradation of the IRE-BP.
