Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

Abstract

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...

DNA and mRNA elements with complementary responses to hemin, antioxidant inducers, and iron control ferritin-L expression

“…3C) and, with the IRE, gave equal responses to FAC or hemin (Fig. 3D), confirming earlier observations that hemin inhibits IRE͞IRP interactions (25) and that FAC and hemin both increase ferritin synthesis (24). The response to hemin of the combined MARE͞ ARE and IRE regulatory elements (Fig.…”

Non-heme Induction of Heme Oxygenase-1 Does Not Alter Cellular Iron Metabolism

“…Interestingly, although we were able to recover the IRE binding activity of IRP1 following hemin treatment by inhibiting heme oxygenase activity, IRP2 was not recoverable. These results are consistent with recent evidence that hemin may inhibit IRP2 activity independently of iron (49,50), although the physiological relevance of this effect is questionable (51), especially in light of the fact that the pool of "uncommitted" heme in non-erythroid tissues is so minute.…”

Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase.

“…In contrast, transferrin receptor mRNA, which contains five highly related IREs in its 3' UTR (Casey et al, 1988), is stabilized by the IRE-IRF interaction, and therefore receptor synthesis is enhanced (Miilner and Kuhn, 1988;Muillner et al, 1989). Highaffinity IRE binding by IRF is induced only under low-iron conditions and is inactivated by high cellular iron levels (Haile et al, 1989;Miillner et al, 1989). The factor is therefore considered to sense a free cellular iron pool and to exert its effects on RNA accordingly.…”