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.     

Xenopus liver ferritin H subunit: cDNA sequence and mRNA production in the liver following estrogen treatment

In vitro translation of liver mRNA from estrogen-treated Xenopus frogs yields two abundant polypeptides in the range of 20 kDa. DNA clones for one of these translation products were isolated and shown to be complementary to mRNA for the heavy subunit of ferritin. The predicted Xenopus amino acid sequence shares about 86% identity with the ferritin heavy chain from bullfrogs and about 70% identity with the comparable mammalian and avian proteins. Clone identity was confirmed by hybridization selection followed by in vitro translation into translation products of 19.5-20 kDa. The nearly full-length cDNA clone, termed XlferH1, comprises 868 nucleotides plus 22 adenosines of the poly(A) tail, including 134 nucleotides of the 5'-untranslated region, a 528-base coding region for 176 amino acids, and a 206-nucleotide 3'-untranslated region. The clone lacks 22 nucleotides from the 5' end of the mRNA. The level of ferritin mRNA in the liver of estrogen-treated frogs was determined over time. The amount of this mRNA relative to total RNA decreased about 3-fold 14 days after estradiol-17 beta was administered. However, the hormone also elevated total RNA in the liver about 24-fold. Hence, the total ferritin mRNA content of the liver increased to about 8 times its initial amount. This pattern of gene expression was very similar to that for serum retinol binding protein. The estrogen induction of these two mRNAs appeared to parallel the overall stimulation of hepatic RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.     

Cloning and expression of a ferritin subunit for Galleria mellonella

Ferritin was purified from iron-fed Galleria mellonella hemolymph by ultra centrifugation and FPLC (Superose 6). SDS-PAGE revealed three bands of 26, 30, and 32 kDa. The ferritin 26 kDa subunit cDNA was obtained from RT-PCR using primer designed from N-terminal sequence analysis. 5'-RACE was used to obtain the complete protein coding sequence. The sequence encodes a 211 amino acid polypeptide including a 20 amino acid leader peptide. An IRE (iron-responsive element) sequence with a predicted stem-loop structure was present in the 5'-UTR of ferritin mRNA. Sequence alignment has a sequence identity with Calpodes ethlius (S)(74%), Drosophila melanogaster (50%), and Aedes aegypti (39%). Northern blot analysis indicated that there were 1.5- and 1.75-fold increases in the expression of ferritin mRNA after iron-fed fat body and midgut, respectively. Also, we confirmed that the ferritin mRNA is not expressed in adult ovary and testis. Arch.     

Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy-terminal tail and identification of the protein using an anti-peptide antibody

The complete sequence of a Dictyostelium discoideum cDNA is presented that codes for monoubiquitin extended at its C-terminus by a 52 amino acid tail. The sequence of both the ubiquitin portion and the tail is highly homologous to the one of Saccharomyces cerevisiae and to a partial mouse sequence. The highly basic tail sequence contains a putative metal and nucleic acid-binding motif. The gene encoding the 0.6 kb mRNA of the C-terminally extended ubiquitin is represented only once in the haploid genome. The 0.6 kb mRNA as well as its translation product, a 15 kDa protein, is expressed in exponentially growing cells and remains present for at least 5 h of development. Using antibodies against a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension was also detected in yeast.     

In vivo and in vitro modulation of the mRNA-binding activity of iron-regulatory factor. Tissue distribution and effects of cell proliferation, iron levels and redox state.

The mRNA-binding protein, iron-regulatory factor (IRF) has a central role in iron metabolism. It coordinately increases transferrin-receptor mRNA stability and inhibits translation of ferritin and erythroid delta-aminolevulinate synthase mRNA by binding to specific mRNA structures, the iron-responsive elements (IRE). In gel-retardation assays, IRF had a broad tissue distribution, showing activity in cytosolic extracts from 12 mouse organs tested. In all these extracts, IRF could be further activated in vitro by 2-mercaptoethanol. In cultured mouse 3T6 fibroblasts, growth stimulation after low serum arrest increased IRF activity 10-fold, mainly through activation of existing inactive IRF. No change was observed during progression of 3T6 cells through the cell cycle. IRF activation by iron chelators has been postulated to result in the reduction of an intramolecular sulfhydryl group. In a search for redox conditions that regulate IRE binding of IRF, we studied several compounds in vitro or in vivo. Hemin, known to inactivate IRF in vivo, showed a similar, reversible effect in vitro, presumably by oxidizing IRF. However, this did not appear to be relevant for the mode of IRF regulation in vivo. Addition of protoporphyrin IX to intact cells induced IRF activity almost to the same extent as desferrioxamine. This effect was inhibited by iron salts, indicating that IRF is activated in vivo through depletion of a chelatable iron pool. In vitro activation by reductants other than 2-mercaptoethanol suggested some selectivity in their access to relevant sulfhydryl groups, but did not reveal which natural redox-sensitive compound might regulate IRF in vivo. However, in cultured cells, inactivation of free IRF by the sulfhydryl-specific oxidizing agent diamide was much more rapidly reversed than inactivation by iron salts. This indicates the direct involvement of a cellular reductant in setting IRF activity and suggests a rate-limiting IRF conformation that is reached only in the presence of iron, but not after diamide oxidation.     

Structure and expression of the cAMP cell-surface receptor

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.     

Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.

CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20-specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2-terminal signal peptide and contains a highly charged COOH-terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

Identification of a rat liver cDNA and mRNA coding for the 28 kDa gap junction protein

By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino-terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A)+ RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3-fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions.     

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.