The inhibition of the iron responsive element RNA-protein interaction by heme does not mimic in vivo iron regulation

Hemin at greater than 1 microM concentrations inhibits the interaction of the iron responsive element (IRE) and the iron responsive element binding protein (IRE-BP) as measured by gel retardation and UV cross-linking. Heme has recently been proposed to inhibit the repression of translation of an IRE-containing mRNA (Lin, J. J., Daniels-McQueen, S., Patino, M. M., Gaffield, L., Walden, W. E., and Thach, R. E., (1990) Science 247, 74-76). Our binding inhibition provides structural support for these observations. The action of hemin, however, does not mimic the physiologically demonstrated inhibition of high affinity binding of the IRE to IRE-BP by the oxidation of a sulfhydryl of the IRE-BP. In addition to this effect, hemin also inhibits a wide variety of RNA and DNA binding proteins, restriction endonucleases, and nucleases. Therefore, in vitro, the inhibitory effects of hemin are not limited to the interaction of the IRE-BP and the IRE, but are nonspecific and affect a wide variety of nucleic acid-protein interactions. Any hypothesis on the effects on protein-nucleic acid interactions employing greater than 1 microM concentrations of hemin should be interpreted with caution.     

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

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.     

Mutagenesis of the iron-regulatory element further defines a role for RNA secondary structure in the regulation of ferritin and transferrin receptor expression.

Within the 5'-untranslated region of ferritin mRNAs, there is a conserved region of 28 nucleotides (nt) (the iron regulatory element (IRE)) that binds a protein (the IRE-binding protein (IRE-BP)) involved in the iron regulation of ferritin mRNA translation. We have examined the role of RNA secondary structure on the interaction of the IRE with the IRE-BP. First, the rat light ferritin IRE possesses a structure similar to that of the bullfrog heavy ferritin IRE (Wang, Y.-H., Sczekan, S. R., and Theil, E. C. (1990) Nucleic Acids Res. 18, 4463-4468). This includes an extended stem, interrupted at various points by bulge nucleotides and a 6-nt single-stranded loop (CAGUGU) at its top. Computer predictions and mapping results suggest the presence of a 3-nt (UGC) bulge 5 bases 5' of the loop in the rat IRE. Second, disruption of the base pairing in the upper stem alters IRE secondary structure and reduces the affinity with which the IRE-BP binds the IRE. Third, increasing the size of the loop or the distance between the UGC bulge and the loop reduces the IRE/IRE-BP interaction. Our results indicate that several aspects of IRE secondary structure are important for its high affinity binding to the IRE-BP.     

The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.     

The hairpin loop but not the bulged C of the iron responsive element is essential for high affinity binding to iron regulatory protein-1

Vertebrates control intracellular iron concentration principally through the interaction of iron regulatory proteins with mRNAs that contain an iron responsive element, a small hairpin with a bulged C. The hairpin loop and bulged C have previously been assumed to be critical for binding and have been proposed to make direct contact with the iron regulatory proteins. However, we show here that a U or G can be substituted for the bulged C provided that specific nucleotides are also present within internal loops. The K(d), IC(50) and chemical modifications of the iron responsive element variants are similar to the wild-type. Results are more consistent with a role in which the C-bulge functions to orient the hairpin for optimal protein binding rather than to directly contact the protein. Characterization of these novel iron responsive element variants may facilitate the identification of additional mRNAs whose expression is controlled by iron regulatory proteins, as well as provide insight into the nature of a critical RNA-protein interaction.     

Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA.

The 5' end of porcine mitochondrial aconitase mRNA contains an iron responsive element (IRE)-like secondary structure (T. Dandekar, R. Stripecke, N. K. Gray, B. Goosen, A. Constable, H. E. Johansson, and M. W. Hentze (1991) EMBO J. 10, 1903-1909). A protein from a liver extract binds to a mitochondrial aconitase RNA probe and supports the identification of this sequence as an IRE. Purified cytosolic aconitase but not the mitochondrial enzyme binds to this IRE as well as to a ferritin IRE. All forms of cytosolic aconitase, [4Fe-4S] enzyme, [3Fe-4S] enzyme and apoenzyme bind with similar affinity. A Kd of 0.25 nM was calculated for the apoaconitase-IRE interaction from Scatchard analysis. These results support the conclusion that cytosolic aconitase is an IRE-binding protein which may regulate translation of mitochondrial aconitase mRNA.     

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60–100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90–156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.     

The importance of a single G in the hairpin loop of the iron responsive element (IRE) in ferritin mRNA for structure: an NMR spectroscopy study.

Noncoding sequences regulate the function of mRNA and DNA. In animal mRNAs, iron responsive elements (IREs) regulate the synthesis of proteins for iron storage, uptake and red cell heme formation. Folding of the IRE was indicated previously by reactivity with chemical and enzymatic probes. 1H- and 31P-NMR spectra now confirm the IRE folding; an atypical 31P-spectrum, differential accessibility of imino protons to solvents, multiple long-range NOEs and heat stable subdomains were observed. Biphasic hyperchromic transitions occurred (52 and 73 degrees C). A G-C base pair occurs in the hairpin loop (HL) (based on dimethylsulfate, RNAse T1 previously used, and changes in NMR imino proton resonances typical of G-C base pairs after G/A substitution). Mutation of the hairpin loop also decreased temperature stability and changed the 31P-NMR spectrum; regulation and protein (IRP) binding were previously shown to change. Alteration of IRE structure shown by NMR spectroscopy, occurred at temperatures used in studies of IRE function, explaining loss of IRP binding. The effect of the HL mutation on the IRE emphasizes the importance of HL structure in other mRNAs, viral RNAs (e.g. HIV-TAR), and ribozymes.