Nitric oxide (NO) produced from L‐arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post‐transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non‐macrophage cell lines activates RNA binding by iron regulatory factor (IRFs), the central trans regulator of mRNAs involved in cellular iron metabolism. NO‐induced binding of IRF to iron‐responsive elements (IRE) specifically represses the translation of transfected IRE‐containing indicator mRNAs as well as the biosynthesis of the cellular iron storage protein ferritin. These findings define a new biological function of NO and identify a regulatory connection between the NO/NOS pathway and cellular iron metabolism.

Several cellular mRNAs are regulated posttranscriptionally by iron-responsive elements (IREs) and the cytosolic IRE-binding proteins IRP-1 and IRP-2. Three different signals are known to elicit IRP-1 activity and thus regulate IRE-containing mRNAs: iron deficiency, nitric oxide (NO), and the reactive oxygen intermediate hydrogen peroxide (H2O2). In this report, we characterize the pathways for IRP-1 regulation by NO and H2O2 and examine their effects on IRP-2. We show that the responses of IRP-1 and IRP-2 to NO remarkably resemble those elicited by iron deficiency: IRP-1 induction by NO and by iron deficiency is slow and posttranslational, while IRP-2 induction by these inductive signals is slow and requires de novo protein synthesis. In contrast, H2O2 induces a rapid posttranslational activation which is limited to IRP-1. Removal of the inductive signal H2O2 after < or = 15 min of treatment (induction phase) permits a complete IRP-1 activation within 60 min (execution phase) which is sustained for several hours. This contrasts with the IRP-1 activation pathway by NO and iron depletion, in which NO-releasing drugs or iron chelators need to be present during the entire activation phase. Finally, we demonstrate that biologically synthesized NO regulates the expression of IRE-containing mRNAs in target cells by passive diffusion and that oxidative stress endogenously generated by pharmacological modulation of the mitochondrial respiratory chain activates IRP-1, underscoring the physiological significance of NO and reactive oxygen intermediates as regulators of cellular iron metabolism. We discuss models to explain the activation pathways of IRP-1 and IRP-2. In particular, we suggest the possibility that NO affects iron availability rather than the iron-sulfur cluster of IRP-1.

Iron‐responsive elements (IREs) are regulatory RNA elements which are characterized by a phylogenetically defined sequence‐structure motif. Their biological function is to provide a specific binding site for the IRE‐binding protein (IRE‐BP). Iron starvation of cells induces high affinity binding of the cytoplasmic IRE‐BP to an IRE which has at least two different known biological consequences, repression of ferritin mRNA translation and stabilization of the transferrin receptor transcript. We report the identification of a novel, evolutionarily conserved IRE motif in the 5′ UTR of murine and human erythroid‐specific delta‐aminolevulinic acid synthase (eALAS) mRNA which encodes the first, and possibly rate limiting, enzyme of the heme biosynthetic pathway. We demonstrate the function of the eALAS IRE as a specific binding site for the IRE‐BP by gel retardation analyses and by in vitro translation experiments. In addition, we show that the 5′ UTR of eALAS mRNA is sufficient to mediate iron‐dependent translational regulation in vivo. These findings strongly suggest involvement of the IRE‐IRE‐BP system in the control of heme biosynthesis during erythroid differentiation.

Communicated by W.I.MattajTranslation of ferritin and erythroid 5-aminolevulinate synthase (eALAS) mRNAs is regulated by iron via mRNA-protein interactions between iron-responsive elements (IREs) and iron regulatory protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated regions of ferritin and eALAS mRNAs and represses translation initiation. The molecular mechanism underlying this translational repression was investigated using reconstituted, IRE-IRP-regulated, cell-free translation systems. The IRE-IRP interaction is shown to prevent the association of the 43S translation pre-initiation complex (including the small ribosomal subunit) with the mRNA. Studies with the spliceosomal protein UlA and mRNAs which harbour specific binding sites for this protein in place of an IRE furthermore reveal that the 5' termini of mRNAs are generally sensitive to repressor proteinmediated inhibition of 43S pre-initiation complex binding.

SummaryThe final assembly of the protein synthesis machinery occurs during translation initiation. This delicate process involves both ends of eukaryotic messenger RNAs as well as multiple sequential protein-RNA and protein-protein interactions. As is expected from its critical position in the gene expression pathway between the transcriptome and the proteome, translation initiation is a selective and highly regulated process. This synopsis summarises the current status of the field and identifies intriguing open questions.

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...

At least two groups of eukaryotic mRNAs (ferritin and erythroid 5-aminolevulinate synthase) are translationally regulated via iron-responsive elements (IREs) located in a conserved position within the 5' untranslated regions of their mRNAs. We establish that the spacing between the 5' terminus of an mRNA and the IRE determines the potential of the IRE to mediate iron-dependent translational repression. The length of the RNA spacer rather than its nucleotide sequence or predicted secondary structure is shown to be the primary determinant of IRE function. When the position of the IRE is preserved, sequences flanking the IRE in natural ferritin mRNA can be replaced by altered flanking sequences without affecting the regulatory function of the IRE in vivo. These results define position as a critical cis requirement for IRE function in vivo and imply the potential to utilize transcription start site selection to modulate the function of this translational regulator.The biosynthesis of the iron storage protein ferritin and of erythroid 5-aminolevulinic acid synthase (eALAS), an enzyme involved in the major iron utilization pathway of the human body, is translationally determined by cellular iron status (1, 6, 7, 8a, 15, 17, 28, 33, 34, 40; unpublished observations). Previous work established that the interaction between an iron-responsive element (IRE) contained in the 5' untranslated region (UTR) of an mRNA and a specific cytoplasmic IRE-binding protein, IRE-BP, results in translational repression of the mRNA in vivo and in vitro (2, 4, 9, 36). Iron regulation of mRNA translation results from irondependent control of the binding activity of 18,24).The presence of an IRE in the 5' UTR of ferritin mRNA has been established as a necessary cis requirement for translational control of ferritin expression (4, 15). However, the conditions under which the presence of an IRE in the 5' UTR of an mRNA suffices for iron control are less clearly defined. When the complete 5' UTR of a ferritin cDNA was fused to human growth hormone (hGH) or chloramphenicol acetyltransferase protein-coding regions or when a synthetic oligodeoxyribonucleotide encoding a ferritin IRE was cloned into the 5' UTR of the hGH or chloramphenicol acetyltransferase gene, expression of the indicator proteins was rendered iron responsive, suggesting that the presence of an IRE in the 5' UTR was sufficient for regulation (1,9,15,17). However, introduction of one or more nonfunctional IRE mutAhts between the 5' end and the intact IRE of an hGH indicator construct renders hGH expression unresponsive to iron (9). On the basis of this finding, the posltion of the IRE was implicated as a possible critical deterhinant of IRE function. Apart from positional requirements, the regions flanking the IRE in ferritin mRNA also seemed to be functionally important (13,38). Since this suggestion was derived from in vitro studies of IRE-BP binding to ferritin mRNA, we evaluated the contribution of the flanking regions to IRE function as a translational regulator in vivo.In thi...

The interaction of ferritin mRNA is regulated by iron via the interaction of a cytoplasmic binding protein (IRE‐BP) with a specific stem‐loop structure in the 5′ untranslated region (UTR), referred to as the iron‐responsive element (IRE). A high affinity RNA‐protein complex between the IRE and the IRE‐BP functions as a repressor of translation in vivo. Translational repression appears to depend upon the position of the IRE in the 5′ UTR of the mRNA. IREs located in the 5′ untranslated region 67 nucleotides or more downstream of the 5′ terminus of the mRNA fail to mediate iron‐dependent translational regulation and give rise to constitutively derepressed transcripts. A model is proposed in which translational regulation of protein biosynthesis involves a position‐dependent interference of the IRE/IRE‐BP complex with one of the initial steps in translation initiation.