Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.

Murray, Mary T.; White, Kristin; Munro, Hamish N.

doi:10.1073/pnas.84.21.7438

Cited by 94 publications

(29 citation statements)

References 37 publications

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“…1A) were prepared as follows: a, "5'-LRNA," a 118-base sense transcript prepared from Sma I-digested pGL-66, a rat L-subunit pseudogene (10), containing the first 65 bases of the 5' UTR (including the conserved sequence), 33 bases of the 5' flanking sequence, and 20 bases from pGEM2; b, "RLFL-RNA," a 198-base sense transcript prepared from HincII-digested rat L-subunit cDNA (19) containing the next 120 bases of the 5' UTR and the first 63 bases of the coding region with 15 bases from spP64; c, "LCOD-RNA," a 247-base sense transcript prepared from Nco I-digested rat L-subunit cDNA (19) containing 227 bases of coding region and 20 bases from pGEM-blue; d, "L3'UTR-RNA," a 190-base sense transcript prepared from HindIII-digested rat L-subunit cDNA (19) containing 148 bases of 3' UTR and 42 bases from pGEM2; e, "L66AS-RNA," a 159-base antisense transcript prepared from Stu I-digested pG-L66, a rat L-subunit pseudogene (10), containing 149 bases of 3' UTR and 10 bases from pGEM2; and also "pGEM-RNA," a 172-base transcript prepared from the Riboprobe Positive Control Template (Promega Biotec, Madison, WI) by using SP6 polymerase. "5'-HRNA" is a 166-base sense transcript containing the 28-base conserved sequence prepared from Dde I-digested pG-H234, a subclone of the rat H-subunit gene (11) [32P]5'-LRNA (Fig. 1B, lane 1), addition of S100 extract retarded its mobility in the form of a smear (Fig.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs.

Leibold

Munro

1988

Proc. Natl. Acad. Sci. U.S.A.

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The mRNAs for the heavy and light subunits of the iron-storage protein ferritin occur in cells largely as inactive ribonucleoprotein particles, which are recruited for translation when iron enters the cell. Cytoplasmic extracts from rat tissues and hepatoma cells were shown by an electrophoretic separation procedure to form RNA-protein complexes involving a highly conserved sequence in the 5' untranslated region of both ferritin heavy-and light-subunit mRNAs. The pattern of complex formation was affected by pretreatment of rats or cells with iron. Crosslinking by UV irradiation showed that the complexes contained an 87-kDa protein interacting with the conserved sequence of the ferritin mRNA. We propose that intracellular iron levels regulate ferritin synthesis by causing changes in specific protein binding to the conserved sequence in the ferritin heavy-and light-subunit mRNAs.

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Section: Methodsmentioning

confidence: 99%

Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs.

Leibold

Munro

1988

Proc. Natl. Acad. Sci. U.S.A.

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652

308

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“…Total RNA or poly(A) ϩ RNA, purified from total RNA using an Oligotex purification system (Qiagen, Valencia, CA), was resolved on 0.9% (poly(A) ϩ RNA) or 1.2% (total RNA) agarose formaldehyde gels. RNA was transferred to a nylon membrane and hybridized with the following 32 P-labeled DNA probes: human TfR, 38 porcine m-Aco, 39 rat FtH subunit, 40 rat FtL pseudogene 66, 35 human 18S rRNA, mouse skeletal muscle ␤-actin (Stratagene, La Jolla, CA), and an IRP1 mouse/human hybrid. 41 Blots were prehybridized for one hour, hybridized overnight with 1 to 1.25 ϫ 10 6 cpm/mL of the indicated probe, washed, and subjected to autoradiography.…”

Section: Northern Analysis and Preparation Of Rnamentioning

confidence: 99%

Effects of iron regulatory protein regulation on iron homeostasis during hypoxia

Schneider

Leibold

2003

Blood

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IntroductionPeriods of low O 2 concentration or hypoxia occur during a broad range of biologic conditions from early development to tumorigenesis and heart disease. Under such situations, cells adapt to the low O 2 environment by increasing the expression of a number of genes including vascular endothelial growth factor, erythropoietin, glycolytic enzymes, 1 and genes involved in iron homeostasis, such as ceruloplasmin, transferrin (Tf), and transferrin receptor (TfR). [2][3][4][5] The expression of these genes requires activation by hypoxia-inducible factor-1 (HIF-1), a transcription factor consisting of hypoxia-inducible HIF-1␣ and constitutively expressed HIF-1␤ subunits. During normoxia, HIF-1␣ is destabilized by a mechanism involving prolyl hydroxylation and targeted for proteasomal degradation. 6 During hypoxia, prolyl hydroxylase activity is reduced and the nonhydroxylated form of HIF-1␣ is stabilized. HIF-1␣ then binds to the constitutively expressed HIF-1␤ subunit to activate transcription of genes that allow for adaptation to hypoxia.When the O 2 concentration returns to normal, the production of toxic reactive oxygen species (ROS), such as the hydroxyl radical ( ⅐ OH), superoxide, and hydrogen peroxide (H 2 O 2 ) increases. Iron contributes to ROS formation by catalyzing the generation of ⅐ OH from H 2 O 2 by Fenton chemistry. 7 ROS, especially ⅐ OH, can damage proteins, DNA, and lipids, and are thought to be responsible for much of the cellular and tissue injury associated with reperfusion disorders. 8,9 In both animal and cell culture models, iron chelation has been shown to decrease the damage caused during reperfusion. [10][11][12] Due to the dual nature of iron as essential for both cellular growth and survival, yet toxic when present in excess, cells have evolved a mechanism to maintain iron homeostasis via iron regulatory protein 1 (IRP1) and IRP2. [13][14][15] When the cellular-free iron pool is low, IRPs bind to specific RNA stem loop structures, called iron-responsive elements (IREs), located in the 5Ј-untranslated region (UTR) of mRNAs such as ferritin heavy chain (FtH) and ferritin light chain (FtL) subunits and mitochondrial aconitase (m-Aco), thereby preventing their translation. IRPs also bind to IREs located in the 3Ј-UTR of TfR mRNA, stabilizing the message from endonucleolytic cleavage and increasing the uptake of Tf-bound iron. Conversely, when the cellular free iron pool is high, IRP2 is degraded by the proteasome in an iron-dependent manner, and IRP1 is converted from an RNA-binding protein to a [4Fe-4S] cluster-containing protein that displays cytosolic aconitase (c-Aco) activity. These changes result in a decrease in TfR synthesis with a corresponding increase in Ft translation, leading to a decrease in the cellular-free iron pool. By constantly responding to changes in the cellular-free iron pool, IRPs maintain iron homeostasis by regulating iron uptake and sequestration.In addition to iron, IRP activities are influenced by other effectors, including ROS and reactive nitrogen s...

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“…In the absence of extracellular iron, a 90-kDa ferritin repressor protein (FRP) binds to a 28-nucleotide iron-responsive element (IRE) in the 5' untranslated region of the ferritin mRNA (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). This prevents translation by ribosomes (9)(10)(11).…”

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confidence: 99%

Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein.

Lin

Patiño

Gaffield

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. (We refer to this interaction as a "crosslink," without intending to imply knowledge as to its chemical nature.) Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent.[14C]Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H202 is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.Synthesis of ferritins, the iron-storage proteins, is regulated primarily at the translational level (1-8). In the absence of extracellular iron, a 90-kDa ferritin repressor protein (FRP) binds to a 28-nucleotide iron-responsive element (IRE) in the 5' untranslated region of the ferritin mRNA (9-18). This prevents translation by ribosomes (9-11). In the presence of extracellular iron, the FRP is inactivated (19, 20) and translation proceeds uninhibited. Derepression of ferritin synthesis can also be observed in vitro, where hemin acts as a highly specific inducer (21,22 (vol/vol) glycerol, 0.05 mM EDTA, and other components as indicated in the figure legends. These conditions are similar to the preincubation step previously described (21), except that small molecules were first removed from all proteins by Centricon 30 (Amicon) ultrafiltration, the glutathione redox buffer was omitted, and the reaction time was 60 min at 370C (maximum crosslinking was ordinarily achieved in 30-60 min). When excess wheat germ proteins were present, reaction times were varied from 15 min (as shown in Fig. 3) to 60 min in order to detect differential rates of crosslinking to different proteins. No such effects were seen. Reaction products were analyzed by SDS/10%o PAGE and fluorography.The stoichiometry of the crosslinking reaction was determined by excising labeled bands from the gel shown in Fig. 2 and allowing the gel slices to swell overnight in Protosol (NEN), before assay of radioactivity in 3A70 scintillation fluid (RPI, Mount Prospect, IL Fig. 1. It is evident that the amount of 14C associated with FRP increased with hemin concentration and that other proteins present in the reaction mixture, such as glyceraldehyde-3-phosphate dehydrogenase, and carbonic anhydrase, were unreactive with hemin. Inclusion of 2-mercaptoethanol slightly stimulated the crosslinking of hemin to FRP, most notably at 50 ,uM [14C]hemin. In other experiments, dithiothreitol was found to be much more stimulative than 2-mercaptoethanol, although it also promoted the no...

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Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.

Cited by 94 publications

References 37 publications

Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs.

Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs.

Effects of iron regulatory protein regulation on iron homeostasis during hypoxia

Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein.

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