Poly(A) polymerase and the regulation of cytoplasmic polyadenylation.

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.

Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements.

Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex. In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5' untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation.

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3' UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3' UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3' UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

Differential regulation of two related RNA-binding proteins, iron regulatory protein (IRP) and IRPB.

The iron regulatory protein (IRP) is a cytoplasmic RNA-binding protein that regulates cellular iron metabolism at the posttranscriptional level. IRP is an unusual bifunctional molecule: in iron-replete cells it predominantly exists as a 4Fe-4S protein and exhibits aconitase enzymatic activity, whereas apo-IRP prevails in iron-starved cells and binds to iron-responsive elements (IREs), structural motifs within the untranslated regions of mRNAs involved in iron metabolism. A related protein with iron-regulated IRE-binding activity, IRPB, was previously identified in rodent cells. IRE-binding by IRP and IRPB is induced by iron deprivation and nitric oxide (NO). Controversial hypotheses have proposed that the induction of IRE-binding activity by iron results either from de novo synthesis of the apo-protein or from a posttranslational conversion of the Fe-S to the apo-protein form. This prompted a detailed analysis of how iron and NO regulate the RNA-binding activities of IRP and IRPB. We demonstrate that IRP is a relatively stable protein (half-life > 12 h). The induction of IRE-binding does not require de novo protein synthesis but results from conversion of Fe-S IRP to apo-IRP. In contrast, IRPB appears less stable in nonstarved cells (half-life approximately 4-6 h) and must be synthesized de novo following iron starvation. Our results furthermore reveal that two RNA-binding proteins with close structural and functional similarities that respond to the same cellular signals are regulated by predominantly different mechanisms.

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.

Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch.

The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the iron-regulatory factor. Iron-regulatory factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to iron-responsive elements and were immunoprecipitated by iron-regulatory-factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in iron-responsive-element-binding of iron-regulatory factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant iron-regulatory factor is a bifunctional protein; after purification, it binds to iron-responsive elements and represses translation in vitro. Following iron treatment, iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-factor seems to be required for the conversion of the iron-responsive-element binding to the aconitase form of the protein.

Translational control of 5-aminolevulinate synthase mRNA by iron-responsive elements in erythroid cells.

Hemoglobin synthesis in red cells is the major iron utilization pathway in the human body and accounts for > 80% of systemic iron turnover. The first step in erythroid heme biosynthesis is catalyzed by a tissue-specific isoform of 5-aminolevulinate synthase (ALAS). The previous identification of iron-responsive elements in the 5'-untranslated region of human and murine erythroid ALAS mRNA raised the intriguing possibility that eALAS expression might be under iron-dependent translational control. As a consequence, a single post-transcriptional regulatory system could coordinate cellular iron acquisition via the transferrin receptor, storage via ferritin, and utilization via eALAS. We directly demonstrate iron-dependent translational regulation of eALAS mRNA in murine erythroleukemia (MEL) cells. The iron-responsive element motif contained in eALAS mRNA is shown to be sufficient to confer translational control to a reporter mRNA both in transfected MEL cells and in vitro.

Modulation of the RNA-binding activity of a regulatory protein by iron in vitro: switching between enzymatic and genetic function?

The iron-responsive element-binding protein (IRE-BP) is an RNA-binding protein that regulates the expression of several mRNAs in response to availability of cellular iron. The iron-dependent control of IRE-BP activity has been reconstituted in vitro. Incubation of purified IRE-BP with iron salts in the presence of the reducing agent cysteine decreases IRE-BP binding to the cognate RNA element. The specificity of this effect is established by several parameters: (i) the interaction of the spliceosomal protein U1A with its U1 small nuclear RNA target sequence as an internal control is unaffected by iron perturbations, (ii) non-iron metals fail to mimic the iron effect, and (iii) iron chelator activates the IRE-binding activity of IRE-BP and titrates the effect of iron salts. Modulation of IRE-BP activity by chelatable iron is reversible and thus does not involve permanent alterations of the integrity of the protein. These findings accurately mirror the physiological basis for iron regulation of transferrin receptor mRNA stability as well as ferritin and erythroid 5-aminolevulinate synthase mRNA translation in vivo. We discuss these data vis-a-vis the structural homology of IRE-BP with the iron-sulfur protein aconitase and propose a mechanism by which the same cytoplasmic protein serves a dual function as an RNA-binding factor and an enzyme.

Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA.

Iron-responsive elements (IREs) are regulatory RNA elements which are characterized by a phylogenetically defined sequence-structure motif. Their biological function is to provide a specific binding site for the IRE-binding protein (IRE-BP). Iron starvation of cells induces high affinity binding of the cytoplasmic IRE-BP to an IRE which has at least two different known biological consequences, repression of ferritin mRNA translation and stabilization of the transferrin receptor transcript. We report the identification of a novel, evolutionarily conserved IRE motif in the 5' UTR of murine and human erythroid-specific delta-aminolevulinic acid synthase (eALAS) mRNA which encodes the first, and possibly rate limiting, enzyme of the heme biosynthetic pathway. We demonstrate the function of the eALAS IRE as a specific binding site for the IRE-BP by gel retardation analyses and by in vitro translation experiments. In addition, we show that the 5' UTR of eALAS mRNA is sufficient to mediate iron-dependent translational regulation in vivo. These findings strongly suggest involvement of the IRE-IRE-BP system in the control of heme biosynthesis during erythroid differentiation.