Subject(s)
Eukaryotic Cells/metabolism , Protein Biosynthesis/physiology , Animals , Artifacts , Binding Sites/physiology , Genes , Genetic Vectors , Humans , Peptide Initiation Factors/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Viral/metabolism , Reproducibility of Results , Research Design , Ribosomes/metabolismABSTRACT
Viruses utilize the protein synthetic machinery of their host. Nonetheless, certain features of the synthesis of viral proteins are distinct from those of host-cell translation. Examples include internal ribosome entry sites in some viral mRNAs and ribosomal frameshifting during production of retroviral proteins. Viruses often inhibit host translation and/or possess mechanisms leading to preferential synthesis of viral proteins. In addition, a participant in the cellular antiviral response is the enzyme PKR (protein kinase, RNA activated), which is involved in the control of cellular translation. Thus, viruses and host cells wage war on the battlefield of translation. The distinctive features of protein synthesis in virally infected cells provide potential targets for therapeutic intervention. Translation-targeted therapeutics have precedence in antibiotics like tetracycline and erythromycin. Means for discovery of translation-targeted therapeutics for viral disease are discussed.
Subject(s)
Drug Design , Protein Biosynthesis/drug effects , Virus Diseases/drug therapy , Animals , Base Sequence , Frameshifting, Ribosomal , Humans , Mammals/genetics , Molecular Sequence Data , RNA, Viral , Retroviridae/genetics , Ribosomes/metabolismABSTRACT
Expression of the structural proteins of the human immunodeficiency virus type 1 (HIV-1), the human T-cell leukemia virus type I (HTLV-I), and of the transferrin receptor (TfR) mRNA depends on posttranscriptional regulatory mechanisms involving both positive and negative elements. In these systems the presence of elements decreasing mRNA expression have been demonstrated. The regulatory proteins (Rev, Rex or iron response element binding protein IRE-BP) antagonize the effects of the downregulatory elements by interacting directly with specific mRNA sites (Rev responsive element, RRE, Rex responsive element, RXRE, or iron responsive elements, IREs) resulting in stabilization and efficient expression of the corresponding mRNAs. To investigate whether this strategy involves common pathways of mRNA utilization, we have studied expression from hybrid mRNAs that contained these previously identified HIV-1 or TfR instability determinants and the binding sites of the regulatory proteins Rev, Rex and/or IRE-BP. Our results demonstrate that only low levels of these hybrid mRNAs accumulate in the absence of the positive regulatory factors Rev, Rex or IRE-BP. The presence of these factors counteracts the effect of heterologous downregulatory elements resulting in increased accumulation of the hybrid mRNAs. However, while Rev or Rex regulation also resulted in efficient protein expression, the IRE-BP only affected mRNA levels without significantly affecting protein expression, suggesting that the pathways of mRNA stabilization/expression are different in these systems.
Subject(s)
Gene Products, rev/metabolism , Gene Products, rex/metabolism , HIV-1/genetics , Human T-lymphotropic virus 1/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , Receptors, Transferrin/genetics , Deferoxamine/pharmacology , Down-Regulation/physiology , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/physiology , Gene Products, gag/genetics , Gene Products, rev/genetics , Gene Products, rex/genetics , Gene Products, tat/genetics , HeLa Cells , Hemin/pharmacology , Humans , Iron-Regulatory Proteins , RNA, Messenger/genetics , RNA, Viral/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Transfection , Tumor Cells, Cultured , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency VirusABSTRACT
The stability of transferrin receptor (TfR) mRNA is regulated by iron availability. When a human plasma-cytoma cell line (ARH-77) is treated with an iron source (hemin), the TfR mRNA is destabilized and a shorter TfR RNA appears. A similar phenomenon is also observed in mouse fibroblasts expressing a previously characterized iron-regulated human TfR mRNA (TRS-1). In contrast, mouse cells expressing a constitutively unstable human TfR mRNA (TRS-4) display the shorter RNA irrespective of iron treatment. These shorter RNAs found in both the hemin-treated ARH-77 cells and in the mouse fibroblasts are shown to be the result of a truncation within the 3' untranslated regions of the mRNAs. The truncated RNA is generated by an endonuclease, as most clearly evidenced by the detection of the matching 3' endonuclease product. The cleavage site of the human TfR mRNA in the mouse fibroblasts has been mapped to single nucleotide resolution to a single-stranded region near one of the iron-responsive elements contained in the 3' UTR. Site-directed mutagenesis demonstrates that the sequence surrounding the mapped endonuclease cleavage site is required for both iron-regulated mRNA turnover and generation of the truncated degradation intermediate. The TfR mRNA does not undergo poly(A) tail shortening prior to rapid degradation since the length of the poly(A) tail does not decrease during iron-induced destabilization. Moreover, the 3' endonuclease cleavage product is apparently polyadenylated to the same extent as the full-length mRNA.
Subject(s)
Endoribonucleases/metabolism , Gene Expression Regulation, Neoplastic , RNA, Messenger/metabolism , Receptors, Transferrin/genetics , Animals , Base Sequence , DNA Mutational Analysis , Gene Expression Regulation, Neoplastic/drug effects , Hemin/pharmacology , Humans , Mice , Molecular Sequence Data , Nucleic Acid Conformation , Plasmacytoma , Poly A/metabolism , RNA-Binding Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Tumor Cells, CulturedSubject(s)
Iron/metabolism , Animals , Base Sequence , Ferritins/genetics , Ferritins/metabolism , Gene Expression , Homeostasis , Humans , Iron-Regulatory Proteins , Molecular Sequence Data , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Transferrin/genetics , Transferrin/metabolismABSTRACT
To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.
Subject(s)
Ferritins/genetics , Gene Expression Regulation , Iron/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Base Sequence , Binding, Competitive , Homeostasis , Iron-Regulatory Proteins , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotide Probes/chemistry , Protein Biosynthesis , Structure-Activity RelationshipSubject(s)
Iron-Binding Proteins , Iron/metabolism , RNA, Messenger/metabolism , Animals , Base Sequence , Ferritins/genetics , Ferritins/metabolism , Homeostasis , Humans , Iron/toxicity , Iron-Regulatory Proteins , Molecular Sequence Data , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolismABSTRACT
The iron-responsive element binding protein (IRE-BP) interacts with specific sequence/structure motifs (iron-responsive elements) within the mRNAs encoding ferritin and the transferrin receptor and thereby post-transcriptionally regulates the expression of these two proteins involved in cellular iron homeostasis. The activity of the IRE-BP is itself regulated by iron such that when cells are treated with an iron source, the RNA binding activity is decreased. The expression of recombinant human IRE-BP in murine cells has been examined as have the expressions of the endogenous IRE-BP of both human and rabbit cells. In all cases, iron down-modulated the RNA binding activity of the IRE-BP, but in no instance was this decrease in activity accompanied by a decrease in the level of the protein as judged by quantitative Western blots. Moreover, the rate of synthesis of the IRE-BP and its rate of degradation have been found to be unaltered by iron manipulation of cells in culture. Consistent with IRE-BP regulation occurring post-translationally, the iron regulation of its activity was found to be unaffected by cycloheximide. These data are discussed in terms of a model of IRE-BP regulation involving the modification of the protein's iron-sulfur center.
Subject(s)
Iron/pharmacology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Animals , Blotting, Western , Cell Line , Cloning, Molecular , Deferoxamine/pharmacology , Escherichia coli/genetics , Ferritins/genetics , Hemin/pharmacology , Humans , Iron-Regulatory Proteins , Kinetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Mice , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , Rabbits , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.
Subject(s)
Aconitate Hydratase/metabolism , Iron-Sulfur Proteins/metabolism , Iron/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , Animals , Apoproteins/metabolism , Deferoxamine/chemistry , Ferricyanides/chemistry , Ferritins/genetics , Hemin/chemistry , Humans , In Vitro Techniques , Iron-Regulatory Proteins , Mice , Oxidation-Reduction , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Several mechanisms of posttranscriptional gene regulation are involved in regulation of the expression of essential proteins of iron metabolism. Coordinate regulation of ferritin and transferrin receptor expression is produced by binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP) to specific stem-loop structures present in target RNAs. The affinity of this protein for its cognate RNA is regulated by the cell in response to changes in iron availability. The IRE-BP demonstrates a striking level of amino acid sequence identity to the iron-sulfur (Fe-S) protein mitochondrial aconitase. Moreover, the recombinant IRE-BP has aconitase function. The lability of the Fe-S cluster in mitochondrial aconitase has led us to propose that the mechanism by which iron levels are sensed by the IRE-BP involves changes in an Fe-S cluster in the IRE-BP. In this study, we demonstrate that procedures aimed at altering the IRE-BP Fe-S cluster in vitro reciprocally alter the RNA binding and aconitase activity of the IRE-BP. The changes in the RNA binding of the protein produced in vitro appear to match the previously described alterations of the protein in response to iron availability in the cell. Furthermore, iron manipulation of cells correlates with the activation or inactivation of the IRE-BP aconitase activity. The results are consistent with a model for the posttranslational regulation of the IRE-BP in which the Fe-S cluster is altered in response to the availability of intracellular iron and this, in turn, regulates the RNA-binding activity.