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.
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.
The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the iron-regulatory factor. Iron-regulatory factor was expressed in vaccinia-virus-infected HeLa cells (hIRF,,) and in Escherichiu coli (hIRF,,,). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRF,, and hIRF,,, bound specifically to iron-responsive elements and were immunoprecipitated by iron-regulatory-factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRF,,, and hIRF,,, was demonstrated in wheat-germ extract. In addition, hIRF,,, and hIRF,,, were shown to display aconitase activity. Treatment of hIRF,,, and hIRF,,, with FeSO, resulted in a drastic reduction in iron-responsive-element-binding of iron-regulatory factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant ironregulatory factor is a bifunctional protein ; after purification, it binds to iron-responsive elements and represses translation in vitro. Following iron treatment, iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-factor seems to be required for the conversion of the iron-responsive-element binding to the aconitase form of the protein.Iron-regulatory factor (IRF), also referred to as iron-responsive-element-binding protein (IRE-BP), femtin repressor protein (FRP) or P90, is a 98-kDa cytoplasmic protein that serves as the central regulatory factor involved in the posttranscriptional control of the expression of ferritin [S -101 and transferrin receptor [ll-121. IRF exerts its effect by binding to iron-responsive elements (IRE) located in the untranslated regions of the mRNAs for the aforementioned proteins in an iron-sensitive manner [I 3 -151. Recently, the recognition of strong amino acid similarities between IRF and the iron-sulfur proteins isopropylmalate isomerase and mitochondria1 aconitase has focussed much interest on the mechanism by which iron regulates the RNA-binding activity of IRF [16, 171. The originally observed similarities between human IRF and these iron-sulfur proteins were further substantiated after labile Fe-S center in equilibrium with the cellular iron supply. In iron loaded cells, it was suggested to possess a 4Fe-4s cluster which prevents binding to IREs; in iron-starved cells, this cluster is likely to be absent, allowing high affinity binding of IRF to IREs [21-261. In support of this hypothesis, biochemically purified mammalian IRF was shown to display aconitase activity [21] and was found to be identical with cytoplasmic aconitase [24, 2.51. Furthermore, we previously demonstrated that ...
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.
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