A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the ⌬U 6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal.The iron-responsive element (IRE), 1 present in the 5Ј-or 3Ј-noncoding regions of animal mRNAs encoding proteins of iron and oxidative metabolism, regulates synthesis of the encoded proteins posttranscriptionally. Iron regulatory proteins (IRPs) bind to the IREs to inhibit ribosome binding or protect mRNA from ribonuclease cleavage (1-5). The predicted secondary structures of the IRE family are hairpins with a six-nucleotide terminal loop (CAGUGN*, N* ϭ A, C, or U), interrupted by an internal loop/bulge (UGC/C) (ferritin-IRE) or a C-bulge (TfR, eALAS, and m-aconitase IREs), that is generally supported by enzymatic cleavage and chemical probing (6 -8); NMR spectroscopy shows a G-C base pair in the hairpin loop and in the internal loop/bulge (9 -12). Two IRE-binding proteins, IRP1 and IRP2, have a high sequence identity except for a 73-amino acid insertion unique to IRP2, and each of them has 30% sequence identity to m-aconitase; IRP1 can have aconitase activity (13-17). IRP1 and IRP2 binding to IREs in iron-depleted cells is abrogated when iron is in excess, with IRP1 forming an [4Fe-4S] cluster (16 -19), and IRP2 being degraded (14, 20 -22). IRP phosphorylation (23, 24), indicates that IRP functions may be integrated with more general metabolic signals.The significance of two IRPs, apparently equivalent in terms of RNA binding and posttranscriptional regulation, is a puzzle, since exclusivity of IRP1 or IRP2 binding for one or another natural IRE sequence has not yet been observed (25-28). IRP binding specificity for the internal loop/bulge and C-bulge of IREs examined in this study, showed that conversion of the ferritin-IRE internal loop/bulge to a C-bulge, by deletion of a single base U 6 , decreased IRP2 binding ...

Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n ) 28-30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505-1512. IRE function (translation) and physical properties (T m and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen) 2 resistant] and more stable (T m increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T m decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The 1 H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.Awareness of mRNA regulation as a mechanism for controlling gene expression is increasing. The iso-IRE (iron responsive element) 1 family of mRNA regulatory elements recognized by the cognate proteins, iron regulatory proteins (IRPs), is one of the most extensively characterized mRNA regulatory targets. However, knowledge about mRNA regulation is in its infancy compared to understanding of DNA regulation such as hormone response elements recognized by hormone nuclear receptors (1). The ancient nature of the proteins encoded in IRE-containing, animal mRNAs (e.g., aconitase, ferritin), the homology of the IRPs to aconitases, and the recent detection of functional IREs and IRPs in bacteria (2) as well as animals suggest that the IRE/IRP interaction is also ancient, possibly representing regulation in an RNA world.The common structural features of iso-IREs are a hairpin hexaloop (CAGUGX) with a trans-loop GC base pair and a helical stem (3-5). The GC base pair in the hexaloop has a large effect on the stabilization of the overall IRE structure (3,6). A disordered C residue occurs in all IREs (4, 5). In many iso-IREs, the disordered C is a bulge in the helix of the IRE stem (4). In another type of iso-IRE, the disordered C residue is part of a set of four conserved residues [UGC-(16 nucleotides)-C]...

Transgenic mice overexpressing leptin backcrossed to the C57BL/6J genetic background (LepTg) have a lean phenotype, characterized by a 95% reduction in adipose mass; reduced plasma levels of glucose, triglycerides, insulin, and IGF-1; and a 75% decrease in adipocyte size. High-fat diet treatment for 20 wk revealed that, compared with normal mice, the LepTg mice had an increased susceptibility to diet-induced obesity, as demonstrated by their rate of weight gain, higher accumulation of sc white adipose tissue mass, hypertrophy of adipocytes, and normalization of their reduced metabolic parameters. The stromal vascular fraction of white adipose tissue from the LepTg mice was highly cellular and contained cells capable of rapid lipid accumulation in primary cultures. The precipitous diet-induced obesity of the LepTg mice was accompanied with 10-fold and 1.6-fold elevations in insulin and IGF-1, respectively, suggesting that the trophic action of insulin and IGF-1 on the preadipocytes and small adipocytes may have caused them to rapidly differentiate and accumulate triacylglycerol stores. Other contributing factors may involve a shift in insulin sensitivity triggered by hyperleptinemia and a decrease in energy expenditure. These studies demonstrate that a chronic response to hyperleptinemia as in the LepTg mice is a predisposing factor to diet-induced obesity and suggest that individuals who are particularly lean because of increased leptin secretion may develop rapid obesity under conditions of a high-fat diet.

Specific messenger RNA (mRNA) structures, identified in solution and exemplified by a set of noncoding mRNA regulatory elements (IRE) 1 and proteins (IRP) (1), control rates of protein synthesis (mRNA translation) or mRNA stability. The iso-IRE family, and the two related phosphorylatable regulatory proteins IRP1 and IRP2, are found in mRNAs encoding proteins of iron and oxygen homeostasis and constitute a natural set of combinatorial mRNA/protein interactions that give quantitatively different responses to cellular iron and oxygen signals (1, 2).Do mRNA tertiary structures such as the ferritin mRNA IRE form in vivo? Metal coordination complexes (MCs), protein nucleases, and alkylating agents have all been used to analyze mRNA solution structure, but MCs are particularly sensitive to the tertiary structure or shape of the RNA binding site (3-7). MCs have specific geometry and relatively rigid shapes contributed by small organic molecules, coordinated to a metal ion (Fig. 1). If the metal is redox active at physiological conditions, as it is for copper, radical cleavage should occur at the MC/RNA binding site to report on the RNA shape. In solution, Cu-phen binds at RNA loops, bulges, and helix distortions in tRNA, mRNA, and rRNA that are indistinguishable for most alkylating agents and too small for access by protein nucleases (3-7). We now show that Cu-phen, an MC, detects the tertiary structure in ferritin mRNA in HeLa cells selectively distinguishing the wild type IL/B from a mutant IRE. Such results link the IRE loop/bulge structure observed directly in solution (6, 8 -12) with the predictions from physiological effects of iron on ferritin and other mRNAs that use IRE/IRP regulation (1, 2) and lay a foundation for studying the behavior of other mRNA structures at the redox conditions in living cells (13). EXPERIMENTAL PROCEDURES CloningFerritin mRNA Expression Plasmid-Plasmid pcDNA3.1-Del-1DV MycHis (ϩ) (Invitrogen) with sequences deleted between the vector transcription start and HindIII, encoded full-length frog H ferritin mRNA and was derived from 1DV (14) using the HindIII site at the 5Ј end and the EcoRI site at the 3Ј end. pcDNA3.1-Del-1DV/Myc-His (ϩ)-⌬U6 has the same ferritin mRNA insert with deletion of U6 in the IRE, to convert the internal loop/ bulge of the Fer-IRE to a C bulge (11).IRP1 Expression Plasmid-IRP1 was encoded in pcDNA3.1-Del-IRP1/ Myc-HIS (ϩ), which contained the 5Ј-untranslated region and the coding region of human IRP1 DNA from pGEM-hIRF (from the ATCC) (15), amplified by PCR, and inserted into the XhoI and HindIII sites.