Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element

Summary The iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5 untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with aK _d of 20–50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.Abbreviations IRE iron-responsive element - FRP ferritin repressor protein - CAT chloramphenicol acetyltransferase - ORF open reading frame     

Purification of a specific repressor of ferritin mRNA translation from rabbit liver

The synthesis of ferritin is regulated at the translation level in coordination with iron availability. Under conditions of low iron, translation of ferritin mRNA is repressed and the majority of ferritin mRNA is non-polysomal. Upon an increase in iron, translation of ferritin mRNA is derepressed resulting in as much as a 50-100-fold increase in the rate of ferritin synthesis. This regulation is mediated at least in part by a specific translational repressor which binds to a conserved sequence, the iron responsive element, located in the 5'-untranslated region of ferritin mRNA. In this communication we report the purification of such a repressor from rabbit liver. This repressor, which we call the "ferritin repressor protein," has an apparent molecular mass of 90 kDa when analyzed by gel filtration chromatography. It inhibits translation of ferritin mRNA in a highly specific fashion when added to a wheat germ lysate programmed with liver poly(A+) mRNA. In addition, it binds specifically to sequences contained within the first 92 nucleotides of ferritin mRNA, most likely the iron responsive element. Analysis of highly purified repressor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it is composed primarily of a single polypeptide of approximately 90 kDa. Elution of this 90-kDa polypeptide from a sodium dodecyl sulfate gel followed by renaturation and analysis for repressor activity shows that it both binds to the 5'-untranslated region of ferritin mRNA and represses its translation in vitro.     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

Role of RNA secondary structure of the iron-responsive element in translational regulation of ferritin synthesis.

Iron regulates synthesis of the iron storage protein ferritin at the translational level through interaction between a stem-loop structure, the iron-responsive element (IRE), located in the 5'-untranslated region (5'-UTR) of ferritin mRNAs, and a protein, the iron regulatory protein (IRP). The role of IRE secondary structure in translational regulation of ferritin synthesis was explored by introducing ferritin constructs containing mutations in the IRE into Rat-2 fibroblasts. Our in vivo studies demonstrate that size and sequence of the loop within the IRE and the distance and/or spatial relationship of this loop to the bulged nucleotide region closest to the loop must be preserved in order to observe iron-dependent translation of ferritin mRNA. In contrast, changes in nucleotide sequence of the upper stem can be introduced without affecting translational regulation in vivo, as long as a stem can be formed. Our in vivo results suggest that only a very small variation in the affinity of interaction of IRP with IRE can be tolerated in order to maintain iron-dependent regulation of translation.     

Determinants of the interaction between the iron-responsive element-binding protein and its binding site in rat L-ferritin mRNA

Ferritin messenger RNA has been shown to be translationally inactivated by the binding of a cytosolic protein to a 28-nucleotide iron-responsive element (IRE) located in the 5'-untranslated region of the mRNA. This interaction has been studied using quantitative receptor-ligand binding methods with gel retardation and nitrocellulose filter binding assays for the separation of bound complex from free RNA. In competition assays the entire 5'-untranslated region and the isolated IRE bound identically. The specificity of the RNA binding was studied using IRE variants. Two IREs from transferrin receptor mRNA and several variants with single base substitutions in the stem or loop had similar affinities. RNAs which could not form a stem-loop structure bound 1000-fold less well. These studies demonstrate the importance of the RNA conformation and the relative insensitivity of binding to much of the primary sequence. Saturation assays with increasing concentrations of 32P-IRE resulted in a binding hyperbola characteristic of mass action binding to a single class of sites with a KD = 0.09 nM. At 37 degrees C the dissociation rate is 0.04 min-1 (t 1/2 = 17 min). This rate is fast enough to account for the shift of ferritin RNA from the ribonucleoprotein pool to polysomes after rats are injected with iron. Determination of the concentration of the repressor requires accounting for three interconverting pools: free active repressor, mRNA-bound protein, and inactive (low affinity) repressor. Rat liver cytosol has a concentration of free active repressor of about 1 pmol/mg protein. Protein bound to endogenous mRNA can be measured by pretreatment with micrococcal nuclease or by separation with DEAE-Sepharose chromatography; it is present at a level similar to that of the free active protein. Inclusion of high levels of thiol reductants in the binding incubations reduces the inactive or low affinity repressor, forming unstably activated protein which has the same KD as the endogenous active protein; this inactive or low affinity protein is 2-4 times more abundant. A mechanism for iron regulation is proposed which accounts for the kinetics, the multiple protein pools, and the characteristics of the protein in these pools.     

The iron-responsive element is the single element responsible for iron-dependent translational regulation of ferritin biosynthesis. Evidence for function as the binding site for a translational repressor

Ferritin, a cytoplasmic protein critical in iron metabolism, displays iron-dependent regulation of its biosynthetic rate with no corresponding changes in mRNA levels. An iron-responsive element (IRE) has been identified in the 5'-untranslated region (UTR) of the human ferritin heavy chain mRNA which, when placed in the 5'-UTR of heterologous reporter genes, confers iron-dependent translational regulation to the hybrid mRNAs. However, whereas the biosynthetic rate of ferritin in response to changes in iron status exhibits a 30-80-fold range, the apparent ranges observed for reporter gene constructs utilizing chloramphenicol acetyltransferase assays or human growth hormone radioimmunoassays have been much less. A deletion and reconstitution study was undertaken to address the possibility that regions of the ferritin gene and mRNA other than the IRE may be necessary for the production of the full range of iron regulation. Data are presented that demonstrate that the IRE alone is capable of conferring iron-dependent translational regulation of biosynthesis to downstream encoded proteins that is both qualitatively and quantitatively similar to that observed with expression of ferritin itself. Thus, the complete range of iron-dependent translational regulation conferred by the IRE occurs independently of the presence of the ferritin promoter, other regions of the ferritin 5'-UTR, the ferritin coding region, and the ferritin 3'-UTR. Additionally, experiments addressing the translatability in vivo of various ferritin construct mRNAs support the theory that the IRE functions as the binding site for a translational repressor.     

Xenopus liver ferritin H subunit: cDNA sequence and mRNA production in the liver following estrogen treatment

In vitro translation of liver mRNA from estrogen-treated Xenopus frogs yields two abundant polypeptides in the range of 20 kDa. DNA clones for one of these translation products were isolated and shown to be complementary to mRNA for the heavy subunit of ferritin. The predicted Xenopus amino acid sequence shares about 86% identity with the ferritin heavy chain from bullfrogs and about 70% identity with the comparable mammalian and avian proteins. Clone identity was confirmed by hybridization selection followed by in vitro translation into translation products of 19.5-20 kDa. The nearly full-length cDNA clone, termed XlferH1, comprises 868 nucleotides plus 22 adenosines of the poly(A) tail, including 134 nucleotides of the 5'-untranslated region, a 528-base coding region for 176 amino acids, and a 206-nucleotide 3'-untranslated region. The clone lacks 22 nucleotides from the 5' end of the mRNA. The level of ferritin mRNA in the liver of estrogen-treated frogs was determined over time. The amount of this mRNA relative to total RNA decreased about 3-fold 14 days after estradiol-17 beta was administered. However, the hormone also elevated total RNA in the liver about 24-fold. Hence, the total ferritin mRNA content of the liver increased to about 8 times its initial amount. This pattern of gene expression was very similar to that for serum retinol binding protein. The estrogen induction of these two mRNAs appeared to parallel the overall stimulation of hepatic RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of the induction of ferritin synthesis by hemin

We have previously reported that hemin derepresses ferritin mRNA translation in vitro. As noted earlier, pre-incubation of a 90 kDa ferritin repressor protein (FRP) with hemin prevented subsequent repression of ferritin synthesis in a wheat germ extract. The significance of this observation has been investigated further. Evidence is presented here that this inactivation of FRP is temperature dependent. Neither FeCl3, Fe3+ chelated with EDTA, nor protoporphyrin IX caused significant inactivation of FRP under comparable conditions, whereas Zn2(+)-protoporphyrin IX produced an intermediate degree of inhibition. The presence of a glutathione redox buffer (GSB), which was previously shown to minimize non-specific side-effects of hemin, was not necessary for the derepression reaction. Inclusion of mannitol, a free radical scavenger, did not alter the inactivation caused by hemin. Calculation of the expected ratio of hemin monomers to dimers suggests that the active species is the monomer.