Increased efficiency of mRNA 3' end formation: a new genetic mechanism contributing to hereditary thrombophilia.

The G-->A mutation at position 20210 of the prothrombin or coagulation factor II gene (F2) represents a common genetic risk factor for the occurrence of thromboembolic events. This mutation affects the 3'-terminal nucleotide of the 3' untranslated region (UTR) of the mRNA and causes elevated prothrombin plasma concentrations by an unknown mechanism. Here, we show that the mutation does not affect the amount of pre-mRNA, the site of 3' end cleavage or the length of the poly(A) tail of the mature mRNA. Rather, we demonstrate that the physiological F2 3' end cleavage signal is inefficient and that F2 20210 G-->A represents a gain-of-function mutation, causing increased cleavage site recognition, increased 3' end processing and increased mRNA accumulation and protein synthesis. Enhanced mRNA 3' end formation efficiency emerges as a novel principle causing a genetic disorder and explains the role of the F2 20210 G-->A mutation in the pathogenesis of thrombophilia. This work also illustrates the pathophysiologic importance of quantitatively minor aberrations of RNA metabolism.

Translational regulation: versatile mechanisms for metabolic and developmental control.

It has become clear that many vital metabolic circuits and early developmental programs are regulated translationally. Until recently, the mechanisms underlying most of these observations were poorly understood. The past year has witnessed several important advances in the understanding of how the translational apparatus is controlled by different regulatory mechanisms.

ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation.

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3' untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.

Iron regulates nitric oxide synthase activity by controlling nuclear transcription.

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Nitric oxide and the post-transcriptional control of cellular iron traffic.

Nitric oxide (NO) is a small, labile and highly reactive molecule generated in various cells by NO synthases. Several important biological functions are controlled by this messenger, and recent data suggest a novel direct role for NO in post-transcriptional gene regulation mediated by iron regulatory protein (IRP). IRP is a cytoplasmic protein that coordinates cellular iron traffic by binding to iron-responsive elements in mRNAs encoding proteins involved in iron uptake, storage and utilization. NO activates the RNA-binding activity of this protein and in this regard mimics the consequences of iron starvation. Cell biological and biochemical data on the functions of NO and IRP suggest a mechanistic basis for these findings and raise the question of their biological implications.

Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction.

Iron-responsive elements (IREs) are RNA motifs that have been identified within the 5' untranslated region of ferritin messenger RNA and the 3' untranslated region of transferrin receptor mRNA. A single IRE mediates iron-dependent control of ferritin translation, whereas multiple IREs are found in the region of the transferrin receptor mRNA responsible for iron-dependent control of mRNA stability. A cytosolic protein binds in vitro to the IREs of both mRNAs. The IRE-binding protein (IRE-BP) is shown to require free sulfhydryl groups for its specific interaction with the IRE. Treatment of lysates with reducing agents increases the binding activity, whereas agents that block sulfhydryls inhibit binding. Iron starvation, leading to decreased ferritin translation, results in increased binding activity, which is explained by an increase in the fraction of the IRE-BP that is in a fully reduced state.

Identification of the iron-responsive element for the translational regulation of human ferritin mRNA.

Regulated translation of messenger RNA offers an important mechanism for the control of gene expression. The biosynthesis of the intracellular iron storage protein ferritin is translationally regulated by iron. A cis-acting element that is both necessary and sufficient for this translational regulation is present within the 5' nontranslated leader region of the human ferritin H-chain messenger RNA. In this report the iron-responsive element (IRE) was identified by deletional analysis. Moreover, a synthetic oligodeoxynucleotide was shown to be able to transfer iron regulation to a construct that would otherwise not be able to respond to iron. The IRE has been highly conserved and predates the evolutionary segregation between amphibians, birds, and man. The IRE may prove to be useful for the design of translationally regulated expression systems.

Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA.

The human ferritin H chain messenger RNA contains a specific iron-responsive element (IRE) in its 5' untranslated region, which mediates regulation by iron of ferritin translation. An RNA gel retardation assay was used to demonstrate the affinity of a specific cytosolic binding protein for the IRE. A single-base deletion in the IRE eliminated both the interaction of the cytoplasmic protein with the IRE and translational regulation. Thus, the regulatory potential of the IRE correlates with its capacity to specifically interact with proteins. Titration curves of binding activity after treatment of cells with an iron chelator suggest that the factor acts as a repressor of ferritin translation.

Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation.

The biosynthetic rates for both the transferrin receptor (TfR) and ferritin are regulated by iron. An iron-responsive element (IRE) in the 5' untranslated portion of the ferritin messenger RNA (mRNA) mediates iron-dependent control of its translation. In this report the 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels. Deletion studies identified a 678-nucleotide fragment of the TfR complementary DNA that is critical for this iron regulation. Five potential stem-loops that resemble the ferritin IRE are contained within the region critical for TfR regulation. Each of two of the five TfR elements was independently inserted into the 5' untranslated region of an indicator gene transcript. In this location they conferred iron regulation of translation. Thus, an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.

From factors to mechanisms: translation and translational control in eukaryotes.

Biochemical and genetic studies are revealing a network of interactions between eukaryotic translation initiation factors, further refining or redefining perceptions of their function. The notion of translated mRNA as a 'closed-loop' has gained support from the identification of physical and functional interactions between the two mRNA ends and their associated factors. Translational control mechanisms are beginning to unravel in sufficient detail to pinpoint the affected step in the initiation pathway.

Finding the hairpin in the haystack: searching for RNA motifs.

A growing list of examples underscores the roles that regulatory RNA motifs play in controlling the genetic repertoire of cells and developing organisms. Once either an RNA-processing signal, a ribozyme, an element that controls translational or mRNA stability or an RNA localization signal has been identified, it is important to search for other RNA sequences that bear similar regulatory signals. While DNA regulatory elements can often be described by a consensus sequence, RNA signals are frequently composed of a combination of sequence and structure motifs. Here, we discuss the approaches that can be used to identify RNA motifs by searching databases.

Position is the critical determinant for function of iron-responsive elements as translational regulators.

At least two groups of eukaryotic mRNAs (ferritin and erythroid 5-aminolevulinate synthase) are translationally regulated via iron-responsive elements (IREs) located in a conserved position within the 5' untranslated regions of their mRNAs. We establish that the spacing between the 5' terminus of an mRNA and the IRE determines the potential of the IRE to mediate iron-dependent translational repression. The length of the RNA spacer rather than its nucleotide sequence or predicted secondary structure is shown to be the primary determinant of IRE function. When the position of the IRE is preserved, sequences flanking the IRE in natural ferritin mRNA can be replaced by altered flanking sequences without affecting the regulatory function of the IRE in vivo. These results define position as a critical cis requirement for IRE function in vivo and imply the potential to utilize transcription start site selection to modulate the function of this translational regulator.

Nitric oxide and oxidative stress (H2O2) control mammalian iron metabolism by different pathways.

Several cellular mRNAs are regulated posttranscriptionally by iron-responsive elements (IREs) and the cytosolic IRE-binding proteins IRP-1 and IRP-2. Three different signals are known to elicit IRP-1 activity and thus regulate IRE-containing mRNAs: iron deficiency, nitric oxide (NO), and the reactive oxygen intermediate hydrogen peroxide (H2O2). In this report, we characterize the pathways for IRP-1 regulation by NO and H2O2 and examine their effects on IRP-2. We show that the responses of IRP-1 and IRP-2 to NO remarkably resemble those elicited by iron deficiency: IRP-1 induction by NO and by iron deficiency is slow and posttranslational, while IRP-2 induction by these inductive signals is slow and requires de novo protein synthesis. In contrast, H2O2 induces a rapid posttranslational activation which is limited to IRP-1. Removal of the inductive signal H2O2 after < or = 15 min of treatment (induction phase) permits a complete IRP-1 activation within 60 min (execution phase) which is sustained for several hours. This contrasts with the IRP-1 activation pathway by NO and iron depletion, in which NO-releasing drugs or iron chelators need to be present during the entire activation phase. Finally, we demonstrate that biologically synthesized NO regulates the expression of IRE-containing mRNAs in target cells by passive diffusion and that oxidative stress endogenously generated by pharmacological modulation of the mitochondrial respiratory chain activates IRP-1, underscoring the physiological significance of NO and reactive oxygen intermediates as regulators of cellular iron metabolism. We discuss models to explain the activation pathways of IRP-1 and IRP-2. In particular, we suggest the possibility that NO affects iron availability rather than the iron-sulfur cluster of IRP-1.

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

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.

Modulation of ferritin H-chain expression in Friend erythroleukemia cells: transcriptional and translational regulation by hemin.

The mechanisms that regulate the expression of the H chain of the iron storage protein ferritin in Friend erythroleukemia cells (FLCs) after exposure to hemin (ferric protoporphyrin IX), protoporphyrin IX, and ferric ammonium citrate (FAC) have been investigated. Administration of hemin increases the steady-state level of ferritin mRNA about 10-fold and that of ferritin protein expression 20-fold. Experiments with the transcriptional inhibitor actinomycin D and transfection studies demonstrate that the increment in cytoplasmic mRNA content results from enhanced transcription of the ferritin H-chain gene and cannot be attributed to stabilization of preexisting mRNAs. In addition to transcriptional effects, translational regulation induces the recruitment of stored mRNAs into functional polyribosomes after hemin and FAC administration, resulting in a further increase in ferritin synthesis. Administration of protoporphyrin IX to FLCs produces divergent transcriptional and translational effects. It increases transcription but appears to suppress ferritin mRNA translation. FAC treatment increases the mRNA content slightly (about twofold), and the ferritin levels rise about fivefold over the control values. We conclude that in FLCs, hemin induces ferritin H-chain biosynthesis by multiple mechanisms: a transcriptional mechanism exerted also by protoporphyrin IX and a translational one, not displayed by protoporphyrin IX but shared with FAC.

Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

The prothrombin 20209 C-->T mutation in Jewish-Moroccan Caucasians: molecular analysis of gain-of-function of 3' end processing.

BACKGROUND: Mutations of the 3' end mRNA-processing signal of the prothrombin (F2) gene have been reported to cause elevated F2 plasma concentrations, thrombosis, and complications of pregnancy. Whereas the common F2 20210*A mutation is almost exclusively found in Caucasians, the F2 20209*T mutation has been reported in Afro-Americans and Afro-Caribbeans only. PATIENTS AND METHODS: Using LightCycler technology, three unrelated Jewish-Moroccan patients tested for obstetric complications were found to be carriers of the F2 20209*T allele. A detailed molecular analysis was performed to identify the functional impact of this mutation. RESULTS: We report three unrelated women of Jewish-Moroccan origin with a F2 20209*T mutation and fetal loss or infertility. The functional analysis revealed that the F2 20209*T mutation stimulates 3' end processing and up-regulates prothrombin protein expression as assessed by a highly sensitive luminescence-based reporter system. CONCLUSIONS: This is the first report of 20209*T in Caucasians, and functional analysis demonstrates that F2 20209*T falls into a general category of mutations of the F2 gene, which may possibly contribute to thrombophilia and complications of pregnancy by interfering with a tightly balanced architecture of non-canonical F2 3' end formation signals.

Iron regulatory factor--the conductor of cellular iron regulation.

All cells have to adjust uptake, utilization and storage of iron according to the availability and their requirement for this essential metal. Progress in recent years has led to the elucidation of the molecular control mechanisms that co-ordinate the uptake, utilization and storage of iron in mammalian cells and has highlighted the role of a newly-identified regulatory protein, the iron regulatory factor (IRF). IRF is a cytoplasmic protein that senses the intracellular iron level and responds by adjusting its function. When the iron level is low, it binds to so-called 'iron responsive elements' (IREs) contained in the mRNAs encoding proteins involved in iron metabolism and erythroid haem synthesis. When levels of cellular iron rise, IRF converts into the enzyme aconitase and looses its ability to bind to IREs. We discuss both functions of this Janus face protein and describe how its function is controlled by the status of an iron sulphur cluster in the IRF protein. We also speculate about how an IRF-mediated regulation may relate to certain medical disorders.

A model for the structure and functions of iron-responsive elements.

Most eukaryotic cells express two proteins, whose biosynthetic rates are determined by the intracellular iron status. The genes for both these proteins, ferritin and the transferrin receptor (TfR), are regulated at the post-transcriptional level, but by entirely different mechanisms. Ferritin mRNA levels are not affected by acute changes in iron availability. Ferritin biosynthesis is regulated translationally via a defined element contained within the 5' untranslated region (UTR) of the ferritin mRNA. This element has been highly conserved during evolution and has been termed an iron-responsive element (IRE). In contrast to ferritin, the regulation of TfR biosynthesis is mirrored by equivalent changes in TfR mRNA levels. The genetic information for this regulation is mostly located in the region of the gene encoding the 3' UTR of the TfR mRNA. Five elements that closely resemble the ferritin IRE are contained within the region which is critical for TfR regulation. The IRE is suggested to function by forming a specific stem-loop structure that interacts with a transacting factor in an iron-dependent fashion. We present a model that accommodates the mediation of distinct post-transcriptional regulatory phenomena via IREs.