RNA Bandshift Assays Research Articles

Grsf1-induced translation of the SNARE protein Use1 is required for expansion of the erythroid compartment.

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5′UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5′UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5′UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.

Post-transcriptional Modulation of Iron Homeostasis during p53-dependent Growth Arrest

Iron plays an essential role in cell proliferation and is a required cofactor for a number of critical cellular enzymes. In this report we investigate changes in proteins of iron metabolism during p53-mediated replicative arrest. Following the induction of p53 in H1299 lung cancer cells containing a doxycycline-inducible p53, an increase in both H and L subunits of ferritin protein was observed. To determine the mechanism of this effect, we investigated the ability of p53 to regulate ferritin. Real time reverse transcription-PCR demonstrated no difference in levels of ferritin H mRNA in the presence and absence of p53. Because these results suggested that transcriptional mechanisms were not responsible for the p53-dependent increase in ferritin, we tested whether a post-transcriptional mechanism was involved. RNA bandshift assays revealed that induction of p53 decreased iron regulatory protein binding. Consistent with this observation, Western blot analysis revealed a decline in transferrin receptor 1 protein levels following induction of p53. Collectively, these results suggest that p53 may induce cell cycle arrest not only by well described mechanisms involving the induction of cyclin-dependent kinase inhibitors but also by the recruitment of pathways that reduce the availability of intracellular iron.

Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1-7 and LSm2-8 complexes from their constituent components in vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1-7 and LSm2-8. Isolated sub-complexes form ring-like higher order structures. LSm1-7 is assembled and stable in the absence of RNA. LSm1-7 forms ring-like structures very similar to LSm2-8 at the EM level. Our in vitro reconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and an in vivo cellular transport assay.

Modulation of Iron Regulatory Protein-1 by Various Metals

Iron regulatory protein-1 (IRP-1) is known as a cytosolic aconitase and a central regulator of iron (Fe) homeostasis. IRP-1 regulates the expression of Fe metabolism-related proteins by interacting with the Fe-responsive element (IRE) in the untranslated regions of mRNAs of these proteins. However, it is less known whether IRP-1 modulates various non-Fe metals. In the present study, we showed that treatment of homogenously purified IRP-1 with non-Fe metals decreased the affinity to IRE in RNA band shift assays and increased aconitase activity. Non-Fe metals also inhibited 55Fe incorporation into the fourth labile position of the Fe–S cluster of IRP-1. In PLC hepatoma cells, metal loading inactivated binding activity and activated enzyme activity. It also suppressed transferrin receptor mRNA expression in the cells. These results suggest that various non-Fe metals modulate IRP-1 by conversion of the 3Fe–4S apo-form to a [1 non-Fe metal + 3Fe]–4Fe holo-form.

Nucleoside Triphosphatase and RNA Helicase Activities Associated with GB Virus B Nonstructural Protein 3

GB virus B (GBV-B) is a positive-stranded RNA virus that belongs to the Flaviviridae family. This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species). Nonstructural protein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymatic activities: serine protease, nucleoside triphosphatase (NTPase), and RNA helicase. The N-terminal serine protease has been characterized and shown to share similar substrate specificity with the HCV NS3 protease. In this report, a full-length GBV-B NS3 protein was expressed in Escherichia coli and purified to homogeneity. This recombinant protein was shown to possess polynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding activities. Both activities were abolished by a single amino acid substitution, from the Lys (K) residue in the conserved walker motif A (or Ia) “AXXXXGK210S” to an Ala (A), confirming that they are intrinsic to GBV-B NS3. Kinetic parameters (Km and kcat) for hydrolysis of various NTPs or dNTPs were obtained. The dsRNA unwinding activity depends on the presence of divalent metal ions and ATP and requires an RNA duplex substrate with 3′ unpaired regions (RNAs with 5′ unpaired regions only or with blunt ends are not suitable substrates for this enzyme). This indicates that GBV-B NS3 RNA helicase unwinds dsRNA in the 3′ to 5′ direction. Direct interaction of the GBV-B NS3 protein with a single-stranded RNA was established using a gel-based RNA bandshift assay. Finally, a homology model of GBV-B NS3 RNA helicase domain based on the 3-dimensional structure of the HCV NS3 helicase that shows a great similarity in overall structure and surface charge distribution between the two proteins was proposed.

Lack of coordinate control of ferritin and transferrin receptor expression during rat liver regeneration.

Transferrin receptor (TfR) and ferritin, key proteins of cellular iron metabolism, are coordinately and divergently controlled by cytoplasmic proteins (iron regulatory proteins, IRP-1 and IRP-2) that bind to conserved mRNA motifs called iron-responsive elements (IRE). IRP, in response to specific stimuli (low iron levels, growth and stress signals) are activated and prevent TfR mRNA degradation and ferritin mRNA translation by hindering ferritin mRNA binding to polysomes. We previously found that, in regenerating liver, IRP activation was accompanied by increased TfR mRNA levels, but not by reduced ferritin expression. The basis for this unexpected behavior was investigated in the present study. Liver regeneration triggered by carbon tetrachloride (CCl4) stimulated by four- to fivefold the synthesis of both L and H ferritin chains. This increase was accompanied with a transcriptionally regulated twofold rise in the amount of ferritin mRNAs. Moreover, polysome-associated ferritin transcripts were fourfold higher in CCl4-treated animals than in control animals. Because RNA bandshift assays showed a fourfold increase in IRP-2 binding activity after CCl4 administration, activated IRP in regenerating liver seemed unable to prevent ferritin mRNAs binding to polysomes. This was confirmed by direct demonstration in the wheat germ translation system that the efficiency of IRP as a translational repressor of a mRNA bearing an IRE motif in front of a reporter transcript is impaired in CCl4-treated rats in spite of an enhanced IRE-binding capacity. In conclusion, we show for the first time that the paradigm of coordinate and opposite control of ferritin and TfR by IRP is contradicted in liver regeneration. Under these circumstances, growth-dependent signals may activate ferritin gene transcription and at the same time hamper the ability of activated IRP-2 to repress translation of ferritin mRNAs, thus preserving for growing liver cells an essential iron-storage compartment.

Response of Monocyte Iron Regulatory Protein Activity to Inflammation: Abnormal Behavior in Genetic Hemochromatosis

AbstractIn genetic hemochromatosis (GH), iron overload affects mainly parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells. We previously found that RE cells from GH patients had an inappropriately high activity of iron regulatory protein (IRP), the key regulator of intracellular iron homeostasis. Elevated IRP should reflect a reduction of the iron pool, possibly because of a failure to retain iron. A defect in iron handling by RE cells that results in a lack of feedback regulation of intestinal absorption might be the basic abnormality in GH. To further investigate the capacity of iron retention in RE cells of GH patients, we used inflammation as a model system as it is characterized by a block of iron release from macrophages. We analyzed the iron status of RE cells by assaying IRP activity and ferritin content after 4, 8, and 24 hours of incubation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). RNA-bandshift assays showed that in monocytes and macrophages from 16 control subjects, IRP activity was transiently elevated 4 hours after treatment with LPS and IFN-γ but remarkably downregulated thereafter. Treatment with NO donors produced the same effects whereas an inducible Nitric Oxide Synthase (iNOS) inhibitor prevented them, which suggests that the NO pathway was involved. Decreased IRP activity was also found in monocytes from eight patients with inflammation. Interestingly, no late decrease of IRP activity was detected in cytokine-treated RE cells from 12 GH patients. Ferritin content was increased 24 hours after treatment in monocytes from normal subjects but not in monocytes from GH patients. The lack of downregulation of IRP activity under inflammatory conditions seems to confirm that the control of iron release from RE cells is defective in GH.

Response of Monocyte Iron Regulatory Protein Activity to Inflammation: Abnormal Behavior in Genetic Hemochromatosis

In genetic hemochromatosis (GH), iron overload affects mainly parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells. We previously found that RE cells from GH patients had an inappropriately high activity of iron regulatory protein (IRP), the key regulator of intracellular iron homeostasis. Elevated IRP should reflect a reduction of the iron pool, possibly because of a failure to retain iron. A defect in iron handling by RE cells that results in a lack of feedback regulation of intestinal absorption might be the basic abnormality in GH. To further investigate the capacity of iron retention in RE cells of GH patients, we used inflammation as a model system as it is characterized by a block of iron release from macrophages. We analyzed the iron status of RE cells by assaying IRP activity and ferritin content after 4, 8, and 24 hours of incubation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). RNA-bandshift assays showed that in monocytes and macrophages from 16 control subjects, IRP activity was transiently elevated 4 hours after treatment with LPS and IFN-γ but remarkably downregulated thereafter. Treatment with NO donors produced the same effects whereas an inducible Nitric Oxide Synthase (iNOS) inhibitor prevented them, which suggests that the NO pathway was involved. Decreased IRP activity was also found in monocytes from eight patients with inflammation. Interestingly, no late decrease of IRP activity was detected in cytokine-treated RE cells from 12 GH patients. Ferritin content was increased 24 hours after treatment in monocytes from normal subjects but not in monocytes from GH patients. The lack of downregulation of IRP activity under inflammatory conditions seems to confirm that the control of iron release from RE cells is defective in GH.

Interactions between HIV rev and nuclear import and export factors: the rev nuclear localisation signal mediates specific binding to human importin-β

The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can “shuttle” between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-β. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-β did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-α/β heterodimer, the binding of Rev to importin-β was actually blocked by importin-α receptor. Our findings suggest that Rev and importin-α bind ( via an arginine-rich sequence) to a similar region on importin-β. In addition, we show that the complex between Rev and importin-β can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.

Regulation of expression of ferritin H-chain and transferrin receptor by protoporphyrin IX.

The effect of protoporphyrin IX (hemin without iron) on the expression of transferrin receptor and ferritin was investigated in Friend leukemia cells. Cells treated with protoporphyrin IX exhibit enhanced transferrin-receptor expression and markedly reduced ferritin synthesis. Stimulation of transferrin-receptor expression is observed at both the mRNA and protein level. The effect on ferritin synthesis is mediated by translational inhibition of the mRNA, which, in contrast, is transcriptionally stimulated by protoporphyrin IX treatment. The regulation of transferrin receptor and ferritin in response to iron perturbations has been studied extensively and is mediated by the binding of iron-regulatory proteins (IRP) to the iron-responsive elements (IRE) present in the 3' and 5' untranslated regions of the transferrin-receptor and ferritin mRNA, respectively. To elucidate the molecular mechanisms underlying the effects of protoporphyrin IX on ferritin and transferrin-receptor expression, the role of the IRE sequence was investigated both in vivo by transfection experiments, with a construct containing the coding region for the chloramphenicol acetyltransferase (CAT) reporter gene under the translational control of the ferritin IRE, and in vitro by RNA band-shift assays. Whereas, examination of IRP binding to the IRE by in vitro assays suggests an apparent inactivation of IRP by protoporphyrin IX treatment, CAT assays indicate that protoporphyrin IX is able to induce in vivo a translational inhibition similar to that obtained by treatment with the iron chelator Desferal. This observation raises the possibility of different effects on the IRP activity exerted by porphyrin treatment in intact tissue-culture cells and in vitro. We conclude that translation of ferritin mRNA and degradation of transferrin-receptor mRNA are inhibited in intact tissue-culture cells by protoporphyrin IX through a mechanism similar to that exerted by iron chelation, thus involving depletion of the intracellular iron pool. These results can improve the understanding of the regulation of ferritin gene expression in some pathological conditions associated with disturbed heme synthesis.

Induction of ferritin synthesis in ischemic-reperfused rat liver: Analysis of the molecular mechanisms

BACKGROUND & AIMS: Iron may catalyze the production of reactive oxygen species (ROS) during postischemic reoxygenation. Ferritin, a cellular iron storage protein, can either represent a source of iron or perform a cytoprotective action against ROS. The aim of this study was to address the role of ferritin in postischemic reperfusion. METHODS: Transcriptional and posttranscriptional mechanisms controlling ferritin gene expression were studied in reperfused rat livers. RESULTS: Proteolysis reduced ferritin levels 2 hours after reperfusion, but a concomitant increase of synthesis, accompanied by enhanced transcription and accumulation of H and L ferritin subunit messenger RNAs (mRNAs), almost re-established normal ferritin content at 4 hours. Pretreatment with interleukin 1 receptor antagonist (IL-1RA) did not prevent the rise of ferritin mRNAs. RNA bandshift assays showed that the activity of the iron regulatory proteins (IRPs), which control ferritin mRNA translation, declined early after reperfusion and recovered progressively thereafter. Pretreatment with either the antioxidant N-acetyl cysteine or IL-1RA was sufficient to prevent almost completely down-regulation of IRP activity. CONCLUSIONS: Postischemic reperfusion causes degradation of ferritin, possibly increasing iron levels. However, induction of ferritin gene transcription, possibly mediated by ferritin-derived iron and ROS- mediated inactivation of IRP, which allows translation of ferritin mRNAs, counteracts this effect and concurs to reestablish the amount of ferritin, which may thus act to limit reperfusion damage. (Gastroenterology 1997 Sep;113(3):946-53)

Resistance to the Antitumor Agent Gallium Nitrate in Human Leukemic Cells Is Associated with Decreased Gallium/Iron Uptake, Increased Activity of Iron Regulatory Protein-1, and Decreased Ferritin Production

The mechanism of drug resistance to gallium nitrate is not known. Since gallium can be incorporated into ferritin, an iron storage protein that protects cells from iron toxicity, we investigated whether ferritin expression was altered in gallium-resistant (R) CCRF-CEM cells. We found that the ferritin content of R cells was decreased, while heavy chain ferritin mRNA levels and iron regulatory protein-1 (IRP-1) RNA binding activity were increased. IRP-1 protein levels were similar in gallium-sensitive (S) and R cells, indicating that R cells contain a greater proportion of IRP-1 in a high affinity mRNA binding state. 59Fe uptake and transferrin receptor expression were decreased in R cells. In both S and R cells, gallium inhibited cellular 59Fe uptake, increased ferritin mRNA and protein, and decreased IRP-1 binding activity. Gallium uptake by R cells was markedly diminished; however, the sensitivity of R cells to gallium could be restored by increasing their uptake of gallium with excess transferrin. Our results suggest that R cells have developed resistance to gallium by down-regulating their uptake of gallium. In parallel, iron uptake by R cells is also decreased, leading to changes in iron homeostasis. Furthermore, since gallium has divergent effects on iron uptake and ferritin synthesis, its action may also include a direct effect on ferritin mRNA induction and IRP-1 activity.

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

AbstractIn 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.

Specific binding of a 47-kilodalton protein to the 3' untranslated region of rat transition protein 2 messenger ribonucleic acid.

The synthesis of transition protein (TNP) 2, one of the predominant nuclear proteins of mammalian spermatids, was shown to be posttranscriptionally regulated, by storing the untranslated mRNA for about 3-5 days in the cytoplasm of differentiating spermatids. It has been proposed that binding of a cytoplasmic protein to a conserved motif of 8 nucleotides (nt) in the 3' untranslated region (3'UTR) of TNP2 mRNA is involved in this translational control mechanism. In this report, we show that deletion or variation of the conserved 8-nt motif (GCCAT-CAC) in rat TNP2-3'UTR abolishes the capacity of the in vitro-transcribed RNA to reconstitute specific RNA-protein complexes in RNA bandshift assays. Using Northwestern analysis, we identified specific binding to the TNP2-3'UTR of four proteins of 45, 47, 49, and 60 kDa, all of which are stage-specifically regulated in male germ cell differentiation. Deletion of the 8-nt motif in the 3'UTR specifically prevented binding of the 47-kDa protein, the interaction of which is thought to be mediated by the RNA secondary structure. Analysis of the RNA secondary structure revealed that the 8-nt motif is an essential element of a specific stem-loop structure that is predicted for rat wild-type TNP2-3'UTR. Therefore we assume that the 47-kDa protein plays an important role in specific RNA-protein complex formation of rat TNP2-3'UTR that may be a central event in the translational control of rat TNP2 mRNA.

Demonstration and characterization of the iron regulatory protein in human brain.

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that regulate expression of ferritin, erythroid 5-aminolevulinic acid synthase, and transferrin receptor through interaction with conserved RNA stem-loop structures called iron-responsive elements (IREs). Two IRPs (IRP1 and IRP2) have been reported. In the present study we provide evidence for and initial characterization of the IRPs in human brain. Two RNA-protein complexes were obtained by RNA band shift assay on cytoplasmic extracts from human brain. Competition studies indicate that the formations of the RNA-protein complexes are specific to the IRE structure. UV crosslinking of brain cytoplasmic extracts with ferritin IRE RNA transcripts revealed a single RNA-protein complex with a molecular mass of 110 kDa. A single band at 100 kDa was obtained with IRP1 antiserum on western blot analysis of brain cytoplasmic extracts, and a supershift in the RNA-protein complexes was observed with an IRP1 antiserum. Two cDNA clones were isolated from a human brain cDNA library with IRP1 cDNA probes, and both of these cDNA probes recognized a single mRNA species (4.0 kb) from human astrocytoma cells. Purified human brain IRP protein has a molecular mass of approximately 100 kDa and is capable of forming two RNA-protein complexes with ferritin IRE RNA and reacts strongly with IRP1 antiserum. These data indicate that IRP1 is predominant in the adult human brain and, in this tissue, is capable of forming a double IRE/IRP complex. This latter observation suggests the brain IRP undergoes posttranslational modification, the result of which may influence the stability of the IRE/IRP complex.

Post-transcriptional control of increased hepatic catalase gene expression in response to oxidative stress.

Catalase is an important member of the antioxidant network that protects the cell against reactive oxygen species (ROS). We studied catalase gene expression in the liver of rats exposed to oxidative stress induced by the ROS-generating drug nitrofurantoin (NF). Catalase enzymatic activity and content are enhanced by NF treatment. The corresponding increase in the steady state level of the messenger ribonucleic acid (mRNA) occurs without significant changes in transcription and seems therefore controlled post-transcriptionally. Indeed, RNA band-shift assays demonstrated a reduced binding of redox-sensitive cytoplasmic protein(s) to the 3' region of catalase mRNA in NF-treated rats, thus suggesting that the redox state of protein that binds to an antioxidant enzyme mRNA may play a role in the hepatic response to oxidative stress.

Nitric‐Oxide‐Mediated Activation of Iron‐Regulatory Protein Controls Hepatic Iron Metabolism During Acute Inflammation

The molecular regulation of intracellular iron metabolism has been studied in the livers of rats undergoing an acute inflammatory reaction following turpentine injection. Treatment induced an increase in the steady-state level of the transferrin receptor (TfR) mRNA, peaking 18 h after treatment and returning to control levels 24 h after treatment, with no change in TfR gene transcription. RNA band-shift assays documented an activation of the cytoplasmic RNA-binding protein called the iron-regulatory protein (IRP), in parallel with a rise in the amount of TfR transcripts. A 2-3-fold increase in the amount of H and L ferritin subunit mRNAs was found 12-18 h after turpentine treatment. Surprisingly, higher accumulation of ferritin mRNAs did not result in appreciable differences in the liver ferritin content. This might be due to the concomitant rise of IRP activity, which is known to prevent ferritin mRNA translation. The absence of significant changes in the total iron and ferritin contents prompted us to investigate the role of nitric oxide (NO), an inflammatory mediator which is also known to modulate the activity of IRP. Northern-blot analysis showed a marked enhancement in the expression of the inducible form of nitric oxide synthase mRNA in turpentine-treated rats. Furthermore, the activation of IRP and the increase of the TfR mRNA content that occur in turpentine-treated rats were abolished by treatment with N5-nitro-L-arginine, a specific nitric oxide synthase inhibitor. The present data suggest that NO-mediated activation of IRP regulates alterations of hepatic iron homeostasis that occur in acute inflammation.

Induction of Ferritin Synthesis by Oxidative Stress: TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION BY EXPANSION OF THE “FREE” IRON POOL

Ferritin, by regulating the "free" intracellular iron pool, controls iron-catalyzed generation of reactive oxygen species, but its role in oxidative damage is still unclear. We show that ferritin synthesis is significantly stimulated in the liver of rats subjected to oxidative stress by treatment with phorone, a glutathione-depleting drug. RNA-bandshift assays document reduced activity of iron regulatory factor, in particular of IRFB, the cytoplasmic protein that post-transcriptionally controls ferritin mRNA translation. Furthermore, Northern blot analysis shows increased accumulation of H and L subunit mRNAs, and nuclear run-on experiments provide evidence of transcriptional activation. Direct measurements of intracellular free iron levels by EPR indicate that the increased ferritin synthesis can be mediated by an expansion of the free iron pool. An early drop of ferritin content after phorone treatment indicates that part of the iron that fuels the free pool might derive from ferritin degradation. Present data seem to suggest that, under conditions of oxidative stress, liver ferritin can represent either a pro- or an anti-oxidant in a time-dependent manner. In fact, its early degradation contributes to expand the intracellular free iron pool that, later on, activates multiple molecular mechanisms to reconstitute ferritin content, thus limiting the pro-oxidant challenge of iron.

Germ Cell-Specific Proteins Interact with the 3′ Untranslated Regions of Prm-1 and Prm-2 mRNA

The testis-specific mouse protamine genes (Prm-1 and Prm-2) are transcribed in haploid round spermatids, their mRNAs stored as cytoplasmic ribonucleoprotein particles and translated about 1 week later in elongating spermatids. We have compared the in vitro translational efficiencies of deproteinized Prm-1 mRNA isolated from purified populations of germ cells and found that Prm-1 mRNA from round spermatids translates as efficiently as Prm-1 mRNA from elongating spermatids, suggesting that translation of Prm-1 mRNA is normally repressed in round spermatids. Previous studies in transgenic mice have shown that the 3′ UTR of Prm-1 mRNA is necessary and sufficient for its translational control (Braun et al., 1989). In this manuscript, we have used an RNA band shift assay to identify an activity, present in cytoplasmic fractions of meiotic spermatocytes and postmeiotic round spermatids, that binds the 3′ UTRs of both Prm-1 and Prm-2 mRNA. We have used 3′ UTR deletion variants to map the binding site to a 22-nt region within the Prm-1 3′ UTR and to a 20-nt region within the Prm-2 3′ UTR. uv cross-linking of the RNA band shift activities detected with the Prm-1 and Prm-2 3′ UTRs generated the same two RNA/protein complexes of 53 and 55 kDa. The presence of the binding activity in the cell type and subcellular compartment associated with Prm-1 and Prm-2 mRNA storage suggest that the activity may be actively engaged in translational repression of these mRNAs.

Transferrin receptor gene expression during rat liver regeneration. Evidence for post-transcriptional regulation by iron regulatory factorB, a second iron-responsive element-binding protein.

Transferrin receptor (TfR) expression is regulated by iron at the level of mRNA stability through a factor (IRF/IRE-BP) which binds to specific iron-responsive elements (IRE). On the other hand, growth-dependent regulation of TfR expression is generally believed to be transcriptionally controlled. We analyzed the molecular mechanisms that control TfR gene expression at the onset of cell proliferation in vivo during liver regeneration after partial hepatectomy. The amount of TfR mRNA increased considerably after partial hepatectomy while run-on assays did not show significant changes in TfR gene transcription. RNA band-shift assays documented a significant activation of IRF/IRE-BP specific for the faster migrating IRE-protein complex (IRFB). These changes occurred in the absence of modifications of total liver iron concentration but together with a significant decrease of ferritin content. Moreover, when extreme variations of liver iron content were achieved by either chronic iron overload or severe iron deficiency, liver regeneration was unable to influence IRE-binding activity. We conclude that IRF/IRE-BP-mediated post-transcriptional control can fully account for TfR mRNA induction during liver cell proliferation in vivo. IRF/IRE-BP activation in the absence of changes in total tissue iron content might depend either on a drop of iron levels into the regulatory pool or on a relatively iron-independent mechanism specific for the faster migrating complex.