The Acute Box cis-Element in Human Heavy Ferritin mRNA 5′-Untranslated Region Is a Unique Translation Enhancer That Binds Poly(C)-binding Proteins

Abstract

Intracellular levels of the light (L) and heavy (H) ferritin subunits are regulated by iron at the level of message translation via a modulated interaction between the iron regulatory proteins (IRP1 and IRP2) and a 5-untranslated region. Iron-responsive element (IRE). Here we show that iron and interleukin-1␤ (IL-1␤) act synergistically to increase H-and L-ferritin expression in hepatoma cells. A GC-rich cis-element, the acute box (AB), located downstream of the IRE in the H-ferritin mRNA 5-untranslated region, conferred a substantial increase in basal and IL-1␤-stimulated translation over a similar time course to the induction of endogenous ferritin. A scrambled version of the AB was unresponsive to IL-1. Targeted mutation of the AB altered translation; reverse orientation and a deletion of the AB abolished the wild-type stem-loop structure and abrogated translational enhancement, whereas a conservative structural mutant had little effect. Labeled AB transcripts formed specific complexes with hepatoma cell extracts that contained the poly(C)-binding proteins, iso-␣CP1 and -␣CP2, which have well defined roles as translation regulators. Iron influx increased the association of ␣CP1 with ferritin mRNA and decreased the ␣CP2-ferritin mRNA interaction, whereas IL-1␤ reduced the association of ␣CP1 and ␣CP2 with H-ferritin mRNA. In summary, the Hferritin mRNA AB is a key cis-acting translation enhancer that augments H-subunit expression in Hep3B and HepG2 hepatoma cells, in concert with the IRE. The regulated association of H-ferritin mRNA with the poly(C)-binding proteins suggests a novel role for these proteins in ferritin translation and iron homeostasis in human liver.The mechanisms governing the regulation of ferritin mRNA translation are complex, but their elucidation is critical to understanding iron homeostasis. Iron and oxidative stress are known to modulate the first stage of translation of ferritin mRNAs when the 43 S ribosome subunit attaches to the 5Ј cap-specific M 7 GpppN in the 5Ј-UTR 1 of L-and H-ferritin mRNAs (1-4). The iron regulatory proteins (IRP1 and IRP2, iso-IRPs) play a central role in regulating ferritin mRNA translation. During conditions of intracellular iron chelation with desferrioxamine (DesF) and oxidative stress, the IRPs bind with higher affinity to the conserved iron-response element (IRE) RNA stem loop 40 nucleotides (nt) downstream of the 5Ј cap sites of the L-and H-ferritin mRNAs (1). This translational repression event prevents attachment of the small ribosome subunit to the 5Ј cap sites of the L-and H-ferritin mRNAs and inhibits ferritin translation (2). In contrast, after iron influx the iso-IRPs are released from the 5Ј cap IREs, and ferritin translation is no longer inhibited, increasing the cellular iron storage capacity (2, 3). The IRP2 knock-out mouse, which is characterized by unregulated ferritin mRNA translation and ferritin accumulation in neurons and gut epithelial cells in a gene-dose manner, validated these observations in vivo (4). Recently zinc and cadmium were ...

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

“…Under identical conditions, the H-ferritin IRE sequences bound to both IRP1 and IRP2 (Fig. 1D), confirming established RNA gel shift assay data in previous reports (15,26,42). These biotinylated RNA pulldown results showed that APP IRE sequences selectively bound to IRP1 and confirmed the results of our IP-RT-PCR experiments (panel A), showing that that IRP2 did not bind to APP IRE sequences under conditions where IRP2 was readily detected to bind to the canonical H-ferritin IRE (n ϭ 8).…”

“…42) for a full method). Supernatants that maintained abundant endogenous mRNAs were used as a positive control for detecting the presence of both H-ferritin and APP RNA under all conditions (Fig.…”

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…The exact mechanism how this is accomplished is not known yet; however, a recent study suggests that Fe 2ϩ might weaken the IRE-IRP repressor complex in vitro (75). Moreover, it was reported that binding of poly-C binding protein 1 to the acute box cis element within the 5Ј-UTR of the human H-ferritin mRNA facilitates H-ferritin translation when the cytosolic iron concentration is increased (76). These data demonstrate that translational inhibitory RNA elements could be modulated by RNA binding proteins.…”

Alternative ferritin mRNA translation via internal initiation

“…Tumor necrosis factor a (TNFa) stimulates transcription of murine H-ferritin mRNA via NF-kB binding to the enhancer element FER2, located 4.8 kb upstream of the transcription start site (Kwak et al 1995). Other cytokines, such as interleukin 1b (IL-1b) enhance H-ferritin mRNA translation via binding of poly(C)-binding proteins to a GC-rich acute box in the 59 UTR (Thomson et al 2005). Xenobiotics and oxidative stress induce ferritin mRNA transcription via binding of Nrf2/Maf or JunD to antioxidant response elements (AREs) within both H-and L-ferritin genes (Tsuji et al 2000;Hintze and Theil 2005;Tsuji 2005).…”