The biosynthetic rates for both the transferrin receptor (TfR) and ferritin are regulated by iron. An iron-responsive element (IRE) in the 5' untranslated portion of the ferritin messenger RNA (mRNA) mediates iron-dependent control of its translation. In this report the 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels. Deletion studies identified a 678-nucleotide fragment of the TfR complementary DNA that is critical for this iron regulation. Five potential stem-loops that resemble the ferritin IRE are contained within the region critical for TfR regulation. Each of two of the five TfR elements was independently inserted into the 5' untranslated region of an indicator gene transcript. In this location they conferred iron regulation of translation. Thus, an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.
Regulated translation of messenger RNA offers an important mechanism for the control of gene expression. The biosynthesis of the intracellular iron storage protein ferritin is translationally regulated by iron. A cis-acting element that is both necessary and sufficient for this translational regulation is present within the 5' nontranslated leader region of the human ferritin H-chain messenger RNA. In this report the iron-responsive element (IRE) was identified by deletional analysis. Moreover, a synthetic oligodeoxynucleotide was shown to be able to transfer iron regulation to a construct that would otherwise not be able to respond to iron. The IRE has been highly conserved and predates the evolutionary segregation between amphibians, birds, and man. The IRE may prove to be useful for the design of translationally regulated expression systems.
The human ferritin H chain messenger RNA contains a specific iron-responsive element (IRE) in its 5' untranslated region, which mediates regulation by iron of ferritin translation. An RNA gel retardation assay was used to demonstrate the affinity of a specific cytosolic binding protein for the IRE. A single-base deletion in the IRE eliminated both the interaction of the cytoplasmic protein with the IRE and translational regulation. Thus, the regulatory potential of the IRE correlates with its capacity to specifically interact with proteins. Titration curves of binding activity after treatment of cells with an iron chelator suggest that the factor acts as a repressor of ferritin translation.
Ferritin plays a key role in determining the intracellular fate of iron and is highly regulated by the iron status of the cell. We have identified a cis-acting element in the transcribed but nontranslated 5' leader sequence of human ferritin heavy-chain mRNA. In transiently transfected murine fibroblasts, the presence of a 157-nucleotide region of the 5' leader sequence was found to be necessary for iron-dependent regulation of ferritin biosynthesis. Further, this 5' leader region is sufficient to transfer iron-mediated translational control to the expression of a heterologous gene product, chloramphenicol acetyltransferase.Ferritin is the major intracellular repository of iron and exists as a heteropolymer of 24 subunits containing both heavy and light chains (1). It serves to sequester and thereby detoxify iron not otherwise utilized for cellular metabolism (2). A more active role of ferritin has been suggested (3), in which iron sequestration may limit and regulate iron availability for cellular metabolism. Ferritin's key role is highlighted by the following observations. (i) All eukaryotic cells investigated thus far have been shown to express ferritin (4). (ii) Cellular proliferation is dependent on the availability of iron, and ferritin levels are closely regulated during normal proliferation and have been found to be abnormal in various malignancies (5, 6). (iii) Ferritin in the cells lining the duodenal mucosa is likely to regulate intestinal iron absorption. Furthermore, since one ofthe most common autosomal recessive disorders, hereditary hemochromatosis, is characterized by pathologically increased absorption of dietary iron, abnormal regulation of ferritin expression or abnormal ferritin function could play a major role in this disorder (7). Thus, the regulation of ferritin levels by iron is the means by which ferritin can both influence and respond to iron homeostasis.It is therefore important to elucidate the mechanisms by which ferritin gene expression is regulated. Munro and his colleagues (8, 9) suggested a translational regulation of ferritin expression in rat liver, based on two observations: (i) actinomycin D does not inhibit the stimulatory effect of iron on ferritin expression and (ii) cytoplasmic ferritin mRNA shifts from an inactive messenger-ribonucleoprotein pool to translationally active polysomes upon iron induction. Similar results were obtained for reticulocytes and liver of bullfrog tadpoles (10, 11). In further support of translational control of ferritin expression, cytoplasmic mRNA levels were shown to remain constant over a wide range of biosynthetic rates induced by iron (9). The recent cloning of the human ferritin heavy-chain cDNA and gene (12)(13)(14)(15) has allowed us to directly address this question of the mechanism of biosynthetic regulation at a molecular level.Previous work showed that a 7.2-kilobase (kb) HindIII genomic DNA fragment (pUCM11) is actively transcribed in stably transformed and transiently transfected murine B6 fibroblasts (13), giving rise to a...
The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four-to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.Virtually all cells must acquire iron from the environment in order to accomplish a wide range of metabolic processes. The necessity for this nutrient coupled with the severe toxicity associated with excess cellular iron demand a metabolic system that is highly regulated. In higher eucaryotic cells, two well-characterized proteins responsible for the uptake and detoxication of iron are the transferrin receptor and ferritin, respectively. The expression of both of these proteins is highly regulated by iron. Interestingly, the information for this regulation is carried out by the mRNA encoding each of the proteins. The RNA element that provides the target for the regulation by iron of the fate of these two mRNA species was first identified in the 5' untranslated region (UTR) of ferritin (1,5,6). In this mRNA, approximately 30 bases are necessary and sufficient for the ability of the ferritin message to be translationally controlled by changes in intracellular iron. This regulatory motif has been named the iron-responsive element (IRE). Although we have not fully defined the details of the RNA that are required for IRE function, present data suggest that both the structure and sequence of this element are important to its function (2,3,13). All known functional IREs form a moderately stable stem-loop structure. The stem is broken by...
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