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M W Hentze

Publications and source records attributed to M W Hentze.

At least 19 recordsLinked to original sources
PubMed1998

Activation of iron regulatory protein-1 by oxidative stress in vitro.

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-gammaS and GTP-gammaS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.

Adenosine Triphosphate↗
PubMed1998

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.

Codon, Nonsense↗
PubMed1998

Iron-regulatory protein-1 (IRP-1) is highly conserved in two invertebrate species--characterization of IRP-1 homologues in Drosophila melanogaster and Caenorhabditis elegans.

Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, we now identify the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.

Amino Acid Sequence↗
PubMed1998

Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast.

The messenger RNA 3' poly(A) tail critically affects the initiation and control of translation in eukaryotes. By analogy to elements involved in transcription initiation, the poly(A) tail has been described as a 'translational enhancer' that enhances the 'translational promoter' activity of the mRNA 5'-cap structure. Elongation or shortening of the poly(A) tail regulates translation during development. Here we show, using cell-free and in vivo translation analyses in Saccharomyces cerevisiae, that the poly(A) tail can act as an independent 'translational promoter', delivering ribosomes to uncapped mRNAs even if their 5' end is blocked. When mRNAs compete for ribosome binding, neither the cap structure nor the poly(A) tail alone is enough to drive efficient translation, but together they synergize and direct ribosome entry to the 5' end. The cap structure both promotes ribosome recruitment, together with the poly(A) tail, and tethers recruited ribosomes to the 5' end. Correct choice of translation initiation codons and the function of translational regulators acting on the 5' untranslated region are thus ensured by the functional interaction of the poly(A) tail with the cap structure.

Base Sequence↗
PubMed1998

A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo.

RNA-protein interactions are central to many aspects of cellular metabolism, cell differentiation, and development as well as the replication of infectious pathogens. We have devised a versatile, broadly applicable in vivo system for the analysis of RNA-protein interactions in yeast. TRAP (translational repression assay procedure) is based on the translational repression of a reporter mRNA encoding green fluorescent protein by an RNA-binding protein for which a cognate binding site has been introduced into the 5' untranslated region. Because protein binding to the 5' untranslated region can sterically inhibit ribosome association, expression of the cognate binding protein causes significant reduction in the levels of green fluorescent protein fluorescence. By using RNA-protein interactions with affinities in the micromolar to nanomolar range, we demonstrate the specificity of TRAP as well as its ability to recover the cDNA encoding a specific RNA-binding protein, which has been diluted 500,000-fold with unrelated cDNAs, by using fluorescence-activated cell sorting. We suggest that TRAP offers a strategy to clone RNA-binding proteins for which little else than the binding site is known, to delineate RNA sequence requirements for protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA-protein interactions in vivo.

Base Sequence↗
PubMed1998

Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.

Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.

Escherichia coli↗
PubMed1998

The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.

Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.

Animals↗
PubMed1997

mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end.

Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3' untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K and hnRNP E1 into HeLa cells specifically silenced the translation of reporter mRNAs bearing a differentiation control element in their 3' untranslated region. Silenced LOX mRNA in rabbit reticulocytes specifically coimmunoprecipitated with hnRNP K. In a reconstituted cell-free translation system, addition of recombinant hnRNP K and hnRNP E1 recapitulates this regulation via a specific inhibition of 80S ribosome assembly on LOX mRNA. Both proteins can control cap-dependent and internal ribosome entry site-mediated translation by binding to differentiation control elements. Our data suggest a specific cytoplasmic function for hnRNPs as translational regulatory proteins.

Amino Acid Sequence↗
PubMed1997

Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intracellular oxidative stress.

We have studied the responses of iron regulatory protein-1 (IRP-1) to extra- and intracellular sources of reactive oxygen intermediates (ROIs). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron metabolism following its activation by iron deficiency, nitric oxide, and administration of H2O2 or antimycin A, an inhibitor of the respiratory chain (Hentze, M. W., and Kühn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 microM H2O2 suffice for complete IRP-1 activation within 60 min when H2O2 is generated extracellularly at steady-state. By contrast, rapid cellular H2O2 degradation necessitates a 5-10-fold higher bolus dose. To study IRP-1 responses to intracellular oxidative stress, mitochondrial respiration was inhibited with antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4-triazole (to diminish H2O2 degradation); in parallel, 2',7'-dichlorodihydrofluorescein diacetate was used as a redox-sensitive probe to monitor intracellular H2O2 levels by fluorescence-activated cell sorting. Catalase inhibition elevates intracellular H2O2, but surprisingly does not cause concomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase in detectable intracellular H2O2. IRP-1 is thus activated both by extra- and intracellular generation of ROIs. While extracellular H2O2 rapidly activates IRP-1 even without detectable increases in intracellular H2O2, intracellular H2O2 elevation is not sufficient for IRP-1 activation. IRP-1 thus represents a novel example of an H2O2-regulated protein that responds differentially to alterations of extra- and intracellular H2O2 levels. Our data also suggest that a direct attack on the 4Fe-4S cluster of IRP-1 by H2O2 (or an H2O2-derived reactive species) represents an unlikely explanation for IRP-1 activation by oxidative stress.

Acetylcysteine↗
PubMed1997

Pathways for the regulation of macrophage iron metabolism by the anti-inflammatory cytokines IL-4 and IL-13.

Macrophage effector functions are influenced by their iron status and by shifts in the balance between type 1 Th1 and Th2 cells. To elucidate the influence of the Th2 cytokines IL-4 and IL-13 on macrophage iron metabolism, we investigated activated primary mouse macrophages and the murine macrophage cell line J774. Stimulation of J774 cells and primary macrophages with IFN-gamma/LPS activates the RNA binding affinities of iron regulatory protein-1 (IRP-1) and IRP-2 for iron-responsive elements, leading to translational repression of the iron storage protein ferritin. Activation of IRP-1 and IRP-2 is caused by increased formation of nitric oxide (NO) via stimulation of the inducible NO synthase by IFN-gamma/LPS. Treatment of macrophages with IL-4 and/or IL-13 before stimulation with IFN-gamma/LPS suppresses NO formation and IRP activation, with concomitantly enhanced ferritin synthesis despite a small reduction in ferritin heavy chain mRNA levels. The mRNA levels for the membrane receptor for iron uptake, transferrin receptor (TfR), decrease following stimulation with IFN-gamma/LPS, although IRP-mediated stabilization of the TfR mRNA would have been expected. This as yet unidentified proximal inhibitory signal by IFN-gamma/LPS is antagonized by IL-4 and/or IL-13, which leads to increased TfR mRNA expression in an IRP-independent manner. Thus, IL-4 and IL-13 regulate the iron metabolism of activated macrophages by at least two different pathways: first, by opposing NO-mediated IRP activation, thereby increasing ferritin translation; and second, by an IRP-independent augmentation of TfR mRNA expression. We suggest that IL-4 and IL-13 may enhance iron uptake and storage in activated macrophages and thereby contribute to down-regulation of macrophage effector functions.

Animals↗
PubMed1997

Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and ribosome association.

The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.

Animals↗
PubMed1997

Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein-1.

A portion of the 3'UTR of the human transferrin receptor mRNA mediates iron-dependent regulation of mRNA stability. The minimal RNA regulatory region contains three conserved hairpins, so-called iron responsive elements (IREs), that are recognized specifically by iron regulatory proteins (IRPs). The structure of this regulatory region and its complex with IRP-1 was probed using a combination of enzymes and chemicals. The data support the existence of an intrinsic IRE loop structure that is constrained by an internal C-G base pair. This particular structure is one of the determinants required for optimal IRP binding. IRP-1 covers one helical turn of the IRE and protects conserved residues in each of the three IREs: the bulged cytosine and nucleotides in the hairpin loops. Two essential IRP-phosphate contacts were identified by ethylation interference. Three-dimensional modeling of one IRE reveals that IRP-1 contacts several bases and the ribose-phosphate backbone located on one face in the deep groove, but contacts also exist with the shallow groove. A conformational change of the IRE loop mediated by IRP-1 binding was visualized by Pb2+-catalyzed hydrolysis. This effect is dependent on the loop structure and on the nature of the closing base pair. Within the regulatory region of transferrin receptor mRNA, IRP-1 induces reactivity changes in a U-rich hairpin loop that requires the presence of the stem-loop structure located just downstream the endonucleolytic cleavage site identified by Binder et al. (Binder R et al. 1994, EMBO J 13:1969-1980). These results provide indications of the mechanism by which IRP-1 stabilizes the transferrin receptor mRNA under iron depletion conditions.

Base Composition↗
PubMed1996

Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Animals↗
PubMed1996

Conservation of aconitase residues revealed by multiple sequence analysis. Implications for structure/function relationships.

Aconitases have recently regained much attention, because one member of this family, iron regulatory protein-1 (IRP-1), has been found to play a dual role as a cytoplasmic aconitase and a regulatory RNA-binding protein. This finding has highlighted a novel role for Fe-S clusters as post-translational regulatory switches. We have aligned 28 members of the Fe-S isomerase family, identified highly conserved amino acid residues, and integrated this information with data on the crystallographic structure of mammalian mitochondrial aconitase. We propose structural and/or functional roles for the previously unrecognized conserved residues. Our findings illustrate the value of detailed protein sequence analysis when high-resolution crystallographic data are already available.

Aconitate Hydratase↗
PubMed1996

Iron-sulphur clusters as genetic regulatory switches: the bifunctional iron regulatory protein-1.

In the eighties, iron regulatory protein-1 (IRP-1) was identified as a cytoplasmic mRNA-binding protein that regulates vertebrate cell iron metabolism. More recently, IRP-1 was found to represent the functional cytoplasmic homologue of mitochondrial aconitase, a citric acid cycle enzyme. Its two functions are mutually exclusive and depend on the status of an Fe-S cluster: the (cluster-less) apoIRP-1 binds to RNA, while the incorporation of a cubane 4Fe-4S cluster is required for enzymatic activity. Cellular signals including iron levels, nitric oxide and oxidative stress can regulate between the two activities posttranslationally and reversibly via the Fe-S cluster. Recent reports suggest that other regulatory proteins may be controlled by similar mechanisms.

Aconitate Hydratase↗