Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region.

Abstract

In previous studies, we showed that acute administration of iron to intact rats or to rat hepatoma cells in culture induces synthesis of the iron-storage protein ferritin by activating translation of inactive cytoplasmic ferritin mRNAs for both the heavy (H) and the light (L) subunits. In the course of activation, these ferritin mRNAs are recruited onto polysomes. To elucidate the structural features of these mRNAs involved in the translational response to iron, a chimera was constructed from the 5' and 3' untranslated regions (UTRs) of ferritin L subunit mRNA fused to the reading frame of the mRNA of bacterial chloramphenicol acetyltransferase (CAT).This chimera and deletion constructs derived from it were introduced into a rat hepatoma cell line by retrovirus-mediated gene transfer. The complete chimera showed increased CAT activity in response to iron enrichment of the medium, whereas deletion of the rst 67 nucleotides of the 5' UTR, which contain a highly conserved sequence, caused loss of regulation by iron. Whereas cis-acting sequences located in the 5' flanking regions of many genes have been repeatedly implicated in modulating their transcriptional expression, we report here a specific regulatory translational sequence found within the 5' UTR of a eukaryotic mRNA.The iron-storage protein ferritin (Mr 450,000) consists in mammals of two types of subunits, a heavier H (M, 22,000) and a slightly lighter L (Mr 20,000) type. Iron administered to rats (1) or added to rat hepatoma cells in culture (2) rapidly increases synthesis of both subunits by mobilizing inactive ferritin L and H mRNAs onto polysomes. This report explores features of the rat ferritin L mRNA involved in this activation process. The 5' untranslated regions (UTRs) of both ferritin mRNAs are unusually long (210 and 168 nucleotides, respectively, for the L and H subunits of the rat), thus differing from most eukaryotic messages with 5' UTRs commonly of 30-60 nucleotides (3). As determined by computer-aided analysis, the first 75 nucleotides of the 5' UTR of L and H subunits in human, rat, and frog yield putative stem-loop structures with calculated free energies of formation (AG values) of -46.1 kcal/mol for rat L mRNA ( Fig. 1) and -40.3 kcal/mol for rat H mRNA (1 kcal = 4.18 kJ). Within this stem-loop structure there is a highly conserved stretch of 28 nucleotides showing almost perfect homology in both H and L mRNAs so far isolated from human (4, 5), rat (6, 7), chicken (8), and frog (9) compared with overall homology of the complete L and H subunit mRNAs of only about 50% in rat and human. To examine the molecular basis for the translational regulation of ferritin mRNA, notably involvement of the untranslated regions, a chimeric gene containing the 5' and 3' UTRs of the ferritin L subunit mRNA fused to the reading frame and 3' UTR of the bacterial chloramphenicol acetyltransferase (CAT) gene has GU C G AU

Effect of chronic renal failure on Na,K-ATPase alpha 1 and alpha 2 mRNA transcription in rat skeletal muscle.

“…Recently, Hsu and Guidotti (24) also described a divergency between the amount ofthe a2 isoform and the transcriptional regulation of the a2 gene in the skeletal muscles of hypokalemic rats. One possibility for the lack of correlation between the amounts of mRNAs and active enzyme is that the transcripts are not translated (25). Another possibility is that the a2 transcripts are translated and the a2 polypeptides do not mature into functional enzyme due to an insufficient amount ofthe ,B chain (26,27).…”

Ovariectomy and estrogen treatment modulate iron metabolism in rat adipose tissue

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Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

“…The folding energy of the stem loop formed by just the AU 1 and AU 2 regions was Ϫ28.4 Kcal/mol and Ϫ16.5 Kcal/mol for the entire 3Ј HSR region. The potential for stem loop protein recognition by RNA-binding proteins has already been established in other systems such as the iron regulatory element-binding proteins (Aziz and Munro, 1987). To evaluate whether VPF/VEGF mRNA elements can promote gene expression by increasing mRNA stability, we designed luciferase-VPF/VEGF mRNA chimeric constructs under the control of a constitutive cytomegalovirus enhancer/promoter (see Figure 7A).…”