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.     

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.     

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.     

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.     

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.     

Evolution of the iron-responsive element

An RNA hairpin structure referred to as the iron-responsive element (IRE) and iron regulatory proteins (IRPs) are key players in the control of iron metabolism in animal cells. They regulate translation initiation or mRNA stability, and the IRE is found in a variety of mRNAs, such as those encoding ferritin, transferrin receptor (Tfr), erythroid aminolevulinic acid synthase (eALAS), mitochondrial aconitase (mACO), ferroportin, and divalent metal transporter 1 (DMT1). We have studied the evolution of the IRE by considering all mRNAs previously known to be associated with this structure and by computationally examining its occurrence in a large variety of eukaryotic organisms. More than 100 novel sequences together with approximately 50 IREs that were previously reported resulted in a comprehensive view of the phylogenetic distribution of this element. A comparison of the different mRNAs shows that the IREs of eALAS and mACO are found in chordates, those of ferroportin and Tfr1 are found in vertebrates, and the IRE of DMT1 is confined to mammals. In contrast, the IRE of ferritin occurs in a majority of metazoa including lower metazoa such as sponges and Nematostella (sea anemone). These findings suggest that the ferritin IRE represents the ancestral version of this type of translational control and that during the evolution of higher animals the IRE structure was adopted by other genes. On the basis of primary sequence comparison between different organisms, we suggest that some of these IREs developed by "convergent evolution" through stepwise changes in sequence, rather than by recombination events.     

Structure of the 5'' untranslated regulatory region of ferritin mRNA studied in solution.

Ferritin mRNAs are the first eukaryotic mRNAs for which a conserved, translational regulatory sequence has been identified. The sequence of twenty-eight nucleotides, called the IRE (iron regulatory element), is found in the 5'-noncoding region and is required for enhanced translation of ferritin mRNA by excess cellular iron; regulation occurs at initiation. The prediction of secondary structure in the IRE is a hairpin loop. We now report an analysis of the IRE structure in solution studied in natural ferritin mRNAs [H and H'(M) subunits] by primer extension, after modification or cleavage by dimethyl sulfate, RNAases T1 and V1, and the chemical nuclease 1, 10-phenanthroline-copper (OPCu) which cleaves single-stranded and bulged regions of RNA. Overall, the structure in solution of the ferritin mRNA regulatory region is a hairpin loop, with magnesium-sensitive features, in which half the stem is provided by the IRE and half by flanking regions; only secondary structure is conserved in the flanking regions. Predicted bulges or internal loops along the stem were clearly detected by OPCu but were missed by the more bulky probe RNAase T1, indicating the efficacy of OPCu in probing subtle features of RNA structure. Magnesium-dependent deviations from the predicted structure were observed in the stem between the hairpin loop and the bulge at C6. The location of the IRE in relation to the initiator AUG or the cap is variable in different ferritin mRNAs. However, the number of nucleotides in the base-paired flanking regions of known ferritin mRNAs is proportional to the distance of the IRE from the cap and places the secondary/tertiary structure 8-10 nucleotides from the cap where interference with initiation is likely.     

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.     

Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.

The regulation of the synthesis of ferritin and erythroid 5-aminolevulinate synthase in mammalian cells is mediated by the interaction of the iron regulatory factor (IRF) with a specific recognition site, the iron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerevisiae. This comprised two components: a constitutively expressed reporter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Induction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bearing mRNAs could also be demonstrated in cell-free yeast extracts. This work defines a minimal system for IRF/IRE translational regulation in yeast that requires no additional mammalian-specific components, thus providing direct proof that IRF functions as a translational repressor in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation.     

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.