Faculty Opinions recommendation of IRP1 regulates erythropoiesis and systemic iron homeostasis by controlling HIF2α mRNA translation.

Abstract Objectives Intracellular iron homeostasis is tightly regulated in posttranscriptional levels via iron regulatory proteins (IRPs). IRPs bind to the iron-responsive elements (IREs), leading to either mRNA translation or stability. Our recent study demonstrated that iron metabolism is intimately linked with adipose tissue browning and thermogenic activation. However, the role of IRP/IRE interactions in the adipose tissue is poorly understood. We aim to characterize the IRP/IRE interactions in the adipose tissue in terms of depot-specificity and thermogenic potential. Methods To induce adipocyte browning, mice were administrated with beta-3 adrenoceptor agonist CL316243 (CL) for 5 days, and different depots of adipose tissue of epididymal (eWAT), inguinal (iWAT), brown (BAT), and liver were collected. Iron metabolism and thermogenesis were evaluated. To investigate the IRP/IRE binding, electrophoretic mobility shift assay (EMSA) was performed in the cytosolic using the fluorescence-labeled IRE (IR-IRE). To distinguish the IRE binding with IRP1 and 2, the cytosolic fraction from IRP1 and 2 knockout mice were used as positive controls. Results In a normal temperature, the constitutive IRP/IRE binding was found in the BAT, but not in the eWAT and iWAT. In response to CL treatment, iron content and transferrin receptor levels significantly increased in the WAT. Accordingly, the IRE/IRPs binding significantly increased in the CL-treated iWAT. Genetic deletion of IRP1 or 2 poses a marginal impact on constitutively active BAT development, suggesting IRP1 and 2 plays a compensatory role. Unlikely to BAT, the deletion of either IRP1 or 2 failed to induce WAT browning in the IRP1 and 2 knockout mice with CL stimulation. Consistently, both IRE binding to IRP1 and 2 were manifest in the CL treated iWAT, implicating that IRP1 and 2 plays a separate and synergistic function for WAT browning. Conclusions Our study defined the depot-specific iron regulatory metabolism in the adipose tissue using an innovative EMSA method. We demonstrated that, for the first time in our knowledge, IRE binding to both IRP1 and IRP2 is indispensable for the thermogenic activation of WAT, which is distinct from the iron regulatory mechanism found in the BAT. We propose that iron metabolism in the WAT is a novel determinant for WAT browning and thermogenic energy expenditure. Funding Sources None.

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IRP1 regulates erythropoiesis and systemic iron homeostasis by controlling HIF2α mRNA translation

Blood ◽

10.1182/blood-2013-03-492454 ◽

2013 ◽

Vol 122 (9) ◽

pp. 1658-1668 ◽

Cited By ~ 73

Author(s):

Nicole Wilkinson ◽

Kostas Pantopoulos

Keyword(s):

Iron Metabolism ◽

Iron Homeostasis ◽

Mrna Translation ◽

Body Iron ◽

Upstream Regulator ◽

Key Points ◽

Age Dependent

Key Points IRP1 controls HIF2α mRNA translation in vivo and thereby acts as an upstream regulator of Epo expression. IRP1 deficiency leads to age-dependent erythropoietic abnormalities and misregulation of body iron metabolism via the HIF2α/Epo pathway.

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A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106964118 ◽

2021 ◽

Vol 118 (27) ◽

pp. e2106964118

Author(s):

Jiandong Chen ◽

Leann To ◽

Francois de Mets ◽

Xing Luo ◽

Nadim Majdalani ◽

...

Keyword(s):

Small Rna ◽

Messenger Rna ◽

Regulatory Networks ◽

Iron Homeostasis ◽

Mrna Translation ◽

Genetic Screen ◽

Phenotypic Traits ◽

E Coli ◽

Small Regulatory Rnas ◽

Screen System

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3′ to 5′ exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3′end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

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Inappropriately High Iron Regulatory Protein Activity in Monocytes of Patients With Genetic Hemochromatosis

Blood ◽

10.1182/blood.v89.7.2546 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2546-2553 ◽

Cited By ~ 75

Author(s):

Gaetano Cairo ◽

Stefania Recalcati ◽

Giuliana Montosi ◽

Elisa Castrusini ◽

Dario Conte ◽

...

Keyword(s):

Iron Homeostasis ◽

Receptor Gene ◽

Regulatory Protein ◽

Mrna Translation ◽

Iron Regulatory Protein ◽

Iron Storage ◽

Control Subjects ◽

Iron Pool ◽

Genetic Hemochromatosis

Abstract In genetic hemochromatosis (GH), excess iron is deposited in parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells until the later stages of the disease. As iron absorption is inversely related to RE cells stores, a failure of RE to retain iron has been proposed as the basic defect in GH. In RE cells of GH subjects, we examined the activity of iron regulatory protein (IRP), a reliable indicator of the elusive regulatory labile iron pool, which modulates cellular iron homeostasis through control of ferritin (Ft) and transferrin receptor gene expression. RNA-bandshift assays showed a significant increase in IRP activity in monocytes from 16 patients with untreated GH compared with 28 control subjects (1.5-fold) and five patients with secondary hemochromatosis (SH) with similar iron burden (fourfold). In 17 phlebotomy-treated GH patients, IRP activity did not differ from that of control subjects. In both GH and SH monocyte-macrophages, Ft content increased by twofold and the L subunit-rich isoferritin profile was unchanged as compared with controls. IRP activity was still upregulated in vitro in monocyte-derived macrophages of GH subjects but, following manipulations of iron levels, was modulated normally. Therefore, the sustained activity of monocyte IRP found in vivo in monocytes of GH patients is not due to an inherent defect of its control, but is rather the expression of a critical abnormality of iron metabolism, eg, a paradoxical contraction of the regulatory iron pool. By preventing Ft mRNA translation, high IRP activity in monocytes may represent a molecular mechanism contributing to the inadequate Ft accumulation and insufficient RE iron storage in GH.

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Conditional Derepression of Ferritin Synthesis in Cells Expressing a Constitutive IRP1 Mutant

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4638-4651.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4638-4651 ◽

Cited By ~ 37

Author(s):

Jian Wang ◽

Kostas Pantopoulos

Keyword(s):

Iron Homeostasis ◽

Regulatory Protein ◽

Mrna Translation ◽

Cell Types ◽

Mrna Levels ◽

Iron Sulfur Cluster ◽

Cellular Iron ◽

Ferritin Synthesis ◽

Significant Enrichment ◽

In Cells

ABSTRACT Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1C437S in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1C437S in a tetracycline-inducible manner. As expected, IRP1C437S stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1C437S-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis.

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Control of Systemic Iron Homeostasis by the 3’ Iron-Responsive Element of Divalent Metal Transporter 1 in Mice

10.1101/2020.02.20.957779 ◽

2020 ◽

Author(s):

Elisabeth Tybl ◽

Hiromi Gunshin ◽

Sanjay Gupta ◽

Tomasa Barrientos ◽

Michael Bonadonna ◽

...

Keyword(s):

Iron Homeostasis ◽

Mrna Translation ◽

Divalent Metal ◽

Targeted Mutagenesis ◽

List Type ◽

Divalent Metal Transporter 1 ◽

Divalent Metal Transporter ◽

Metal Transporter ◽

Iron Responsive Element ◽

Responsive Element

AbstractDivalent metal transporter 1 (DMT1) is essential for dietary iron assimilation and erythroid iron acquisition. The 3’ untranslated region of the murine DMT1 mRNA contains an iron responsive element (IRE) that is conserved in humans but whose functional role remains unclear. We generated and analyzed mice with targeted disruption of the DMT1 3’IRE. These animals display hypoferremia during the suckling period, associated with a reduction of DMT1 mRNA and protein in the intestine. In contrast, adult mice exhibit hyperferremia, accompanied by enlargement of hepatic and splenic iron stores. Intriguingly, disruption of the DMT1 3’IRE in adult animals augments intestinal DMT1 expression, in part due to increased mRNA translation. Hence, during postnatal growth, the DMT1 3’IRE promotes intestinal DMT1 expression and secures iron sufficiency; in adulthood, it suppresses DMT1 and prevents systemic iron loading. This work demonstrates that the 3’IRE of DMT1 plays a role in the control of DMT1 expression and systemic iron homeostasis, and reveals an age-dependent switch in its activity.Key pointsTargeted mutagenesis of the 3’IRE of DMT1 in mice reveals its importance for maintenance of systemic iron homeostasis.The 3’IRE stimulates intestinal DMT1 expression and prevents hypoferremia during early life, but exerts opposite effects in adulthood

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A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

10.1101/2021.05.11.443692 ◽

2021 ◽

Author(s):

Jiandong Chen ◽

Leann To ◽

Francois de Mets ◽

Xing Luo ◽

Nadim Majdalani ◽

...

Keyword(s):

Small Rna ◽

Regulatory Networks ◽

Iron Homeostasis ◽

Mrna Translation ◽

Genetic Screen ◽

Phenotypic Traits ◽

E Coli ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Screen System

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation as well as virulence. sRNAs can alter target mRNA translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify new regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in E. coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX and McaS, previously found to act through Hfq competition, RNase T, a 3′-5′ exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two new specific regulators were identified. AspX, a novel 3′ end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

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Brain transcriptome analysis reveals subtle effects on mitochondrial function and iron homeostasis of mutations in the SORL1 gene implicated in early onset familial Alzheimer’s disease

10.1101/2020.07.17.207787 ◽

2020 ◽

Cited By ~ 1

Author(s):

Karissa Barthelson ◽

Stephen Martin Pederson ◽

Morgan Newman ◽

Michael Lardelli

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Early Onset ◽

Iron Homeostasis ◽

Mrna Translation ◽

Complete Loss ◽

Loss Of Function ◽

Wild Type ◽

Expression Of Genes ◽

Insight Into

AbstractBackgroundTo prevent or delay the onset of Alzheimer’s disease (AD), we must understand its molecular basis. The great majority of AD cases arise sporadically with a late onset after 65 years of age (LOAD). However, rare familial cases of AD can occur due to dominant mutations in a small number of genes that cause an early onset prior to 65 years of age (EOfAD). As EOfAD and LOAD share similar pathologies and disease progression, analysis of EOfAD genetic models may give insight into both subtypes of AD. Sortilin-related receptor 1 (SORL1) is genetically associated with both EOfAD and LOAD and provides a unique opportunity to investigate the relationships between both forms of AD. Currently, the role of SORL1 mutations in AD pathogenesis is unclear.MethodsTo understand the molecular consequences of SORL1 mutation, we performed targeted mutagenesis of the orthologous gene in zebrafish. We generated an EOfAD-like mutation, V1482Afs, and a putatively null mutation, to investigate whether EOfAD-like mutations in sorl1 display haploinsufficiency by acting through loss-of-function mechanisms. We performed mRNA-sequencing on whole brains comparing normal (wild type) fish with their siblings heterozygous for EOfAD-like or complete loss-of-function mutations in sorl1 or transheterozygous for these mutations. Differential gene expression and gene set enrichment analyses identified, respectively, changes in young adult zebrafish brain transcriptomes, and putative effects on neural subcellular functions.ResultsWe identified subtle effects on expression of genes involved in energy production, mRNA translation and mTORC1 signalling in both the EOfAD-like and null mutant brains, implying that these effects are due to sorl1 haploinsufficiency. Surprisingly, we also observed changes to expression of genes occurring only in the EOfAD-mutation carrier brains, suggesting gain-of-function effects. Transheterozygosity for the EOfAD-like and null mutations (i.e. lacking wild type sorl1), caused apparent effects on iron homeostasis and other transcriptome changes distinct from the single-mutation heterozygous fish.ConclusionsOur results provide insight into the possible early brain molecular effects of an EOfAD mutation in human SORL1. Differential effects of heterozygosity and complete loss of normal SORL1 expression are revealed.

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