Selective Translational Control of the Alzheimer Amyloid Precursor Protein Transcript by Iron Regulatory Protein-1

Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.     

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60–100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90–156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.     

Novel 5′ Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer''s disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer''s disease.     

Excess capacity of the iron regulatory protein system

Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Stable cell lines were created in which either IRP1, IRP2, or both were knocked down. Knockdown of IRP1 decreased IRE binding activity but did not affect ferritin H and transferrin receptor 1 (TfR1) expression, whereas knockdown of IRP2 marginally affected IRE binding activity but caused an increase in ferritin H and a decrease in TfR1. Knockdown of both IRPs resulted in a greater reduction of IRE binding activity and more severe perturbation of ferritin H and TfR1 expression compared with single IRP knockdown. Even though the knockdown of IRP-1, IRP-2, or both was efficient, resulting in nondetectable protein and under 5% of wild type levels of mRNA, all stable knockdowns retained an ability to modulate ferritin H and TfR1 appropriately in response to iron challenge. However, further knockdown of IRPs accomplished by transient transfection of small interfering RNA in stable knockdown cells completely abolished the response of ferritin H and TfR1 to iron challenge, demonstrating an extensive excess capacity of the IRP system.     

Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer's diseased brains.

Iron regulatory proteins (IRPs) are cytoplasmic mRNA binding proteins involved in intracellular regulation of iron homeostasis. IRPs regulate expression of ferritin and transferrin receptor at the mRNA level by interacting with a conserved RNA structure termed the iron-responsive element (IRE). This concordant regulation of transferrin receptors and ferritin is designed so a cell can obtain iron when it is needed, and sequester iron when it is in excess. However, we have reported that iron accumulates in the brain in Alzheimer's disease without a concomitant increase in ferritin. An increase in iron without proper sequestration can increase the vulnerability of cells to oxidative stress. Oxidative stress is a component of many neurological diseases including Alzheimer's. We hypothesized that alterations in the IRP/IRE interaction could be the site at which iron mismanagement occurs in the Alzheimer's brains. In this report we demonstrate that in normal human brain extracts, the IRP is detected as a double IRE/IRP complex by RNA band shift assay, but in 2 of 6 Alzheimer's brain (AD) extracts examined a single IRE/IRP complex was obtained. Furthermore, the mobility of the single IRE/IRP complex in Alzheimer's brain extracts is decreased relative to the double IRE/IRP complex. Western blot and RNA band super shift assay demonstrate that IRP1 is involved in the formation of the single IRE/IRP complex. In vitro analyses suggest that the stability of the doublet complex and single AD complex are different. The single complex from the AD brain are more stable. A more stable IRE/IRP complex in the AD brain could increase stability of the transferrin receptor mRNA and inhibit ferritin synthesis. At the cellular level, the outcome of this alteration in the molecular regulatory mechanism would be increased iron accumulation without an increase in ferritin; identical to the observation we reported in AD brains. The appearance of the single IRE/IRP complex in Alzheimer's brain extracts is associated with relatively high endogenous ribonuclease activity. We propose that elevated RNase activity is one mechanism by which the iron regulatory system becomes dysfunctional.     

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.     

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.     

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.     

Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs.

Translation of ferritin and erythroid 5-aminolevulinate synthase (eALAS) mRNAs is regulated by iron via mRNA-protein interactions between iron-responsive elements (IREs) and iron regulatory protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated regions of ferritin and eALAS mRNAs and represses translation initiation. The molecular mechanism underlying this translational repression was investigated using reconstituted, IRE-IRP-regulated, cell-free translation systems. The IRE-IRP interaction is shown to prevent the association of the 43S translation pre-initiation complex (including the small ribosomal subunit) with the mRNA. Studies with the spliceosomal protein U1A and mRNAs which harbour specific binding sites for this protein in place of an IRE furthermore reveal that the 5' termini of mRNAs are generally sensitive to repressor protein-mediated inhibition of 43S pre-initiation complex binding.     

The cellular mechanisms of body iron homeostasis

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transferrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.     

Regulation of the 75-kDa subunit of mitochondrial complex I by iron

Iron homeostasis is tightly regulated, as cells work to conserve this essential but potentially toxic metal. The translation of many iron proteins is controlled by the binding of two cytoplasmic proteins, iron regulatory protein 1 and 2 (IRP1 and IRP2) to stem loop structures, known as iron-responsive elements (IREs), found in the untranslated regions of their mRNAs. In short, when iron is depleted, IRP1 or IRP2 bind IREs; this decreases the synthesis of proteins involved in iron storage and mitochondrial metabolism (e.g. ferritin and mitochondrial aconitase) and increases the synthesis of those involved in iron uptake (e.g. transferrin receptor). It is likely that more iron-containing proteins have IREs and that other IRPs may exist. One obvious place to search is in Complex I of the mitochondrial respiratory chain, which contains at least 6 iron-sulfur (Fe-S) subunits. Interestingly, in idiopathic Parkinson's disease, iron homeostasis is altered, and Complex I activity is diminished. These findings led us to investigate whether iron status affects the Fe-S subunits of Complex I. We found that the protein levels of the 75-kDa subunit of Complex I were modulated by levels of iron in the cell, whereas mRNA levels were minimally changed. Isolation of a clone of the 75-kDa Fe-S subunit with a more complete 5'-untranslated region sequence revealed a novel IRE-like stem loop sequence. RNA-protein gel shift assays demonstrated that a specific cytoplasmic protein bound the novel IRE and that the binding of the protein was affected by iron status. Western blot analysis and supershift assays showed that this cytosolic protein is neither IRP1 nor IRP2. In addition, ferritin IRE was able to compete for binding with this putative IRP. These results suggest that the 75-kDa Fe-S subunit of mitochondrial Complex I may be regulated by a novel IRE-IRP system.     

Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells

Mammalian cells regulate iron levels tightly through the activity of iron-regulatory proteins (IRPs) that bind to RNA motifs called iron-responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Likewise, intestinal epithelial cells regulate iron absorption by a process that also depends on the intracellular levels of iron. Although intestinal epithelial cells have an active IRE/IRP system, it has not been proven that this system is involved in the regulation of iron absorption in these cells. In this study, we characterized the effect of overexpression of the ferritin IRE on iron absorption by Caco-2 cells, a model of intestinal epithelial cells. Cells overexpressing ferritin IRE had increased levels of ferritin, whereas the levels of the transferrin receptor were decreased. Iron absorption in IRE-transfected cells was deregulated: iron uptake from the apical medium was increased, but the capacity to retain this newly incorporated iron diminished. Cells overexpressing IRE were not able to control iron absorption as a function of intracellular iron, because both iron-deficient cells as well as iron-loaded cells absorbed similarly high levels of iron. The labile iron pool of IRE-transfected cell was extremely low. Likewise, the reduction of the labile iron pool in control cells resulted in cells having increased iron absorption. These results indicate that cells overexpressing IRE do not regulate iron absorption, an effect associated with decreased levels of the regulatory iron pool.