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PubMedDate not supplied

Reversible inhibition of translation by Xenopus oocyte-specific proteins.

A characteristic of growing oocytes of all animal species is the synthesis and accumulation of messenger RNA which is destined to be used primarily by the early embryo. The mechanism(s) which regulates the translation of this maternal mRNA remains unknown. However, the inability of the oocyte to translate all of its putative mRNA has been attributed to at least three limitations: (1) The rate of translation is limited by the availability of components of the translational apparatus other than mRNA, (2) the structural organization of the mRNA prevents translation, and (3) proteins associated with the mRNA prevent translation. Several investigators have suggested that proteins associated with maternal mRNA suppress translation in sea urchin eggs, although others claim that such results may be due to experimental artefacts. Oocyte-specific proteins have been identified in association with non-translating poly(A)+ mRNAs from Xenopus laevis oocytes, and we report here that when these proteins are reconstituted with mRNAs in vitro the translation of the mRNAs in vitro is reversibly repressed. The implication is that these proteins are involved in the regulation of translation of stored maternal mRNAs.

Animals↗

PubMed1999

Identification of TIAR as a protein binding to the translational regulatory AU-rich element of tumor necrosis factor alpha mRNA.

In monocyte/macrophages, the translation of tumor necrosis factor alpha (TNF-alpha) mRNA is tightly regulated. In unstimulated cells, translation of TNF-alpha mRNA is blocked. Upon stimulation with lipopolysaccharides, this repression is overcome, and the mRNA becomes efficiently translated. The key element in this regulation is the AU-rich element (ARE). We have previously reported the binding of two cytosolic protein complexes to the TNF-alpha mRNA ARE. One of these complexes (complex 1) forms with extracts of both unstimulated and lipopolysaccharide-stimulated macrophages and requires a large fragment of the ARE containing clustered AUUUA pentamers. The other complex (complex 2) is only detected after cell activation, binds to a minimal UUAUUUAUU nonamer, and is composed of a 55-kDa protein. Here, we report the identification of the RNA-binding protein TIAR as a protein involved in complex 1. The RNA sequence bound by TIAR and the cytoplasmic localization of this protein in macrophages argue for an involvement of TIAR in TNF mRNA posttranscriptional regulation.

Animals↗

PubMed2001

Repression of Manduca sexta ferritin synthesis by IRP1/IRE interaction.

Mammalian ferritin subunit synthesis is controlled at the translational level by the iron regulatory protein 1 (IRP1)/iron responsive element (IRE) interaction. Insect haemolymph ferritin subunit messages have an IRE in the 5'-untranslated region (UTR). We have shown that recombinant M. sexta IRP1 represses the in vitro translation of both the heavy and light chain ferritin subunits from this species without altering transcription. Deletion of either the 5'-UTR or the IRE from the mRNA abolishes IRP1 repression. Our studies indicated that the translational control of ferritin synthesis by IRP/IRE interaction could occur in insects in a manner similar to that of mammals. To our knowledge, this is the first report of the control of insect ferritin synthesis by IRP1/IRE interaction. Furthermore, this is the first indication that the synthesis of a secreted ferritin subunit can also be controlled in this manner.

Amino Acid Sequence↗

PubMed2002

DNA methyltransferase and demethylase in human prostate cancer.

Recent studies have shown that cytosine-5 methylation at CpG islands in the regulatory sequence of a gene is one of the key mechanisms of inactivation. The enzymes responsible for CpG methylation are DNA methyltransferase (DNMT) 1, DNMT3a, and DNMT3b, and the enzyme responsible for demethylation is DNA demethylase (MBD2). Studies on methylation-demethylation enzymes are lacking in human prostate cancer. We hypothesize that MBD2 enzyme activity is repressed and that DNMT1 enzyme activity is elevated in human prostate cancer. To test this hypothesis, we analyzed enzyme activities, mRNA, and protein levels of MBD2 and DNMT1, DNMT3a, and DNMT3b in human prostate cancer cell lines and tissues. The enzyme activities of DNMTs and MBD2 were analyzed by biochemical assay. The mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction and by Northern blotting. The protein expression was measured by immunohistochemistry with specific antibodies. The results of these experiments demonstrated that (1) the activity of DNMTs was twofold to threefold higher in cancer cell lines and cancer tissues, as compared with a benign prostate epithelium cell line (BPH-1) and benign prostatic hyperplasia (BPH) tissues; (2) MBD2 activity was lacking in prostate cancer cell lines but present in BPH-1 cells; (3) immunohistochemical analyses exhibited higher expression of DNMT1 in all prostate cancer cell lines and cancer tissues, as compared with BPH-1 cell lines and BPH tissues; (4) MBD2 protein expression was significantly higher in BPH-1 cells and lacking in prostate cancer cell lines and, in BPH tissues, MBD2 protein expression was poorly observed, as compared with no expression in prostate cancer tissues; and (5) mRNA expression for DNMT1 was upregulated in prostate cancer, as compared with BPH-1, and mRNA expression for MBD2 was found to be significantly expressed in all cases. The results of these studies clearly demonstrate that DNMT1 activity is upregulated, whereas MBD2 is repressed at the level of translation in human prostate cancer. These results may demonstrate molecular mechanisms of CpG hypermethylation of various genes in prostate cancer.

Cell Line↗

PubMed2005

Plant and animal microRNAs: similarities and differences.

Plant and animal microRNAs (miRNAs) are evolutionarily ancient small RNAs, approximately 19-24 nucleotides in length, that are generated by cleavage from larger highly structured precursor molecules. In both plants and animals, miRNAs posttranscriptionally regulate gene expression through interactions with their target mRNAs, and these targets are often genes involved with regulating key developmental events. Despite these similarities, plant and animal miRNAs exert their control in fundamentally different ways. Generally, animal miRNAs repress gene expression by mediating translational attenuation through (multiple) miRNA-binding sites located within the 3' untranslated region of the target gene. In contrast, almost all plant miRNAs regulate their targets by directing mRNA cleavage at single sites in the coding regions. These and other differences suggest that the two systems may have originated independently, possibly as a prerequisite to the development of complex body plans.

Animals↗

PubMed1989

Prosomes discriminate between mRNA of adenovirus-infected and uninfected HeLa cells.

Prosomes are small cytoplasmic RNP complexes associated with repressed mRNA. In in vitro translation, they discriminate between the mRNA of adenovirus-infected HeLa cells and those of uninfected cells grown under normal conditions. Prosomes as well as their RNA constituents interact much more strongly with poly(A)+ mRNA of infected cells and inhibit their translation in vitro preferentially. A possible role of prosomes in the differential regulation of translation is discussed.

Adenoviridae↗

PubMed1988

Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli.

A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], the vectors carry a strong hybrid trp/lac promoter, the lacZ ribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacIq allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.

Bacterial Proteins↗

PubMed2006

HIV-l and the microRNA-guided silencing pathway: an intricate and multifaceted encounter.

MicroRNAs (miRNAs) are approximately 21-24 nucleotide RNAs that mediate repression of messenger RNA (mRNA) translation through recognition of specific miRNA binding sites usually located in the 3' non-translated region. Designed to simulate miRNAs, small interfering RNAs represent a powerful genetic approach to potently inhibit gene expression by mediating cleavage of the intended mRNA target. This strategy has been applied successfully to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). However, recent evidences indicate that viral RNAs may themselves be processed, to some extent, by the endogenous miRNA biosynthetic machinery in mammalian cells, extending previous observations in plants. The resulting viral miRNAs may exert regulatory effects towards host and/or viral genes that may influence viral replication and modulate the course of infection. Viral miRNA generation and/or action may be limited by counteraction through inhibitory viral RNAs and/or proteins. This review article will focus on the relationship between HIV-1 and miRNA-guided RNA silencing, and discuss the different aspects of their interaction. As we learn more about the mechanism and importance of small RNA-based antiviral systems, a more intricate picture of the interaction between HIV-1 and a proven antiviral defense mechanism in lower eukaryotes is emerging.

Animals↗

PubMed2001

Stability of barley aleurone transcripts: Dependence on protein synthesis, influence of the starchy endosperm and destabilization by GA3.

We have studied the stability of Barley aleurone and embryo expressed (Balem) transcripts in aleurone layers. The Per1, Ole1 and Ole2 transcripts are abundant during desiccation and in dry resting seeds, while B12D and B22E transcripts are expressed mainly during seed maturation and germination. From 21 to 40 days post anthesis (DPA) incubation of aleurone layers resulted in a substantial, but differential reduction in the levels of these transcripts. In contrast, Balem transcript levels in aleurone layers of incubated embryoless grains were (except for B22E) similar to those of freshly dissected layers. Cycloheximide lowered transcript levels significantly. This indicates that a protein-synthesis-dependent mRNA-stabilizing mechanism is active in the aleurone cells when attached to the starchy endosperm. At the onset of seed desiccation (40 DPA), half-lives of transcripts to be stored in the dry seed were up to several days longer than the half-life of B22E, which decreases during seed maturation. While the Per1, Ole1 and Ole2 transcript levels decline rapidly in the aleurone layers of mature, germinating seeds, the genes are actively transcribed and their transcripts highly stable in the aleurone of incubated embryoless seeds. The expression of Ole1 and Ole2, as well as Per1, can be repressed 100-1 000-fold by gibberellic acid (GA3) in a dose-dependent manner. Abscisic acid can counteract the GA3 repression. Incubations with transcriptional and translational inhibitors indicate that GA3 inhibits the transcription of these genes and at the same time induces a protein-synthesis-dependent mechanism destabilizing their mRNA molecules present.

Journal Article↗

PubMed1999

Taming of transposable elements by homology-dependent gene silencing.

Transposable elements can invade virgin genomes within a few generations, after which the elements are 'tamed' and retain only limited transpositional activity. Introduction of the I element, a transposon similar to mammalian LINE elements, into Drosophila melanogaster genomes devoid of such elements initially results in high-frequency transposition of the incoming transposon, high mutation rate, chromosomal nondisjunction and female sterility, a syndrome referred to as hybrid dysgenesis (for review, see refs 2-4); a related syndrome has also been described in mammals. High-frequency transposition is transient, as the number of I elements reaches a finite value and transposition ceases after approximately ten generations. It has been proposed that the I elements encode a factor that negatively regulates their own transcription, but evidence for such a mechanism is lacking. Using the hybrid dysgenesis syndrome in Drosophila as a model, we show here that transpositional activity of the I element can be repressed by prior introduction of transgenes expressing a small internal region of the I element. This autoregulation presents features characteristic of homology-dependent gene silencing, a process known as cosuppression. Repression does not require any translatable sequence, its severity correlates with transgene copy number and it develops in a generation-dependent manner via germline transmission of a silencing effector in females only. These results demonstrate that transposable elements are prone to and can be tamed by homology-dependent gene silencing, a process that may have emerged during the course of evolution as a specific defense mechanism against these elements.

Animals↗

PubMed2001

Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis.

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold-inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a DeltaHik33 mutant using the DNA microarray technique. In wild-type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold-repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold-regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.

Bacterial Proteins↗

PubMed2003

A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis.

Morphogenesis in Saccharomyces cerevisiae and the pathogenic yeast Candida albicans is governed in part by the same molecular circuits. In S. cerevisiae, FLO11/MUC1 expression has been shown to be modulated by multiple signalling pathways required for pseudohyphal development. We have established a screen in S. cerevisiae to identify regulators of fungal development in C. albicans based on FLO11::lacZ expression as a reporter. This screen identified both known components of the mitogen-activated protein kinase (MAPK) cascade and the cAMP cascade that are important for hyphal development in C. albicans, as well as genes not yet known to be involved in morphogenesis. The Candida homologue of MCM1 is one of the novel factors identified in this screen as being important for morphogenesis. CaMcm1p levels do not vary significantly in different cell types and respond to an autoregulatory feedback mechanism, arguing that CaMcm1p activity is regulated by post-translational modifications. Both overexpression and repression of this essential gene led to the induction of hyphae. Moreover, we found that the expression of HWP1, a hyphae-specific gene, was induced by repression of CaMCM1. The changes in morphology and HWP1 expression were not the result of a change in expression levels of NRG1 or TUP1, known repressors of hyphal development. Thus, CaMcm1p is a component of a hitherto unknown regulatory mechanism of hyphal growth.

Base Sequence↗

PubMed1982

Messenger RNA species partially in a repressed state in mouse sarcoma ascites cells.

Four major mRNA species of mouse sarcoma ascites cells, coding for polypeptides designated P65, P40, P36, and P21, occur predominantly as untranslated messenger ribonucleoprotein particles. Cloned cDNA probes were used to study their distribution in cytoplasmic extracts of these cells. A considerable portion of the mRNA molecules sedimented as small particles, whereas the rest was present in polyribosomes. In contrast, the actin mRNA was present almost exclusively in polyribosomes. Incubation of the ascites cells in culture medium, particularly after a starvation treatment, caused an enhancement in polypeptide chain initiation relative to elongation in these cells, as evidenced by a shift of ribosomes into the polyribosome fraction and by an increase in polyribosome size. Exposure of the cells to a low concentration of cycloheximide, an inhibitor of the elongation step, had a similar effect. The actin mRNA and the active P65, P40, P36, and P21 mRNA molecules were shifted to larger polyribosomes in the treated cells, but no shift of molecules from small particles to polyribosomes was observed. The incubation in culture also led to considerable increases in the proportion of P65 and P40 mRNA molecules in the untranslated state. The results indicate that the untranslated state cannot be attributed to poor initiation efficiency. It is suggested that a portion of the mRNA molecules is maintained in a repressed state and that mRNA repression may represent an important translation control process.

Actins↗

PubMed1991

Iron and the liver.

Iron is essential for life, but iron overload is toxic and potentially fatal. The liver is a major site of iron storage and is particularly susceptible to injury from iron overload, especially when (as in primary hemochromatosis) the iron accumulates in hepatocytes. Iron can be taken up by the liver in several forms and by several pathways including: (1) receptor-mediated endocytosis of diferric or monoferric transferrin or ferritin, (2) reduction and carrier-facilitated internalization of iron from transferrin without internalization of the protein moiety of transferrin, (3) electrogenic uptake of low molecular weight, non-protein bound forms of iron, and (4) uptake of heme from heme-albumin, heme-hemopexin, or hemoglobin-haptoglobin complexes. Normally, pathway 2 is probably the major one for uptake of iron by hepatocytes. Iron is stored in the liver in the cores of ferritin shells and as hemosiderin, an insoluble product derived from iron-rich ferritin. Iron in hepatocytes stimulates translation of ferritin mRNA and represses transcription of DNA for transferrin and transferrin receptors. The major pathologic effects of chronic hepatic iron overload are: (1) fibrosis and cirrhosis, (2) porphyria cutanea tarda, and (3) hepatocellular carcinoma. Although precise pathogenetic mechanisms remain unknown, iron probably produces these and other toxic effects by increasing oxidative stress and lysosomal lability. Vigorous efforts at diagnosis and treatment of iron overload are essential since the pathologic effects of iron are totally preventable by early vigorous iron removal and prevention of iron re-accumulation.

Ascorbic Acid↗

PubMed1995

A Bordetella pertussis fepA homologue required for utilization of exogenous ferric enterobactin.

The bfeA (Bordetella ferric enterobactin) receptor gene was cloned from a Bordetella pertussis chromosomal library by using a screen in Escherichia coli to detect iron-repressed genes encoding exported proteins translationally fused to the E. coli phoA gene. The bfeA gene encoded a protein with a molecular mass of approximately 80 kDa and about 50% amino acid sequence identity to both the fepA- and pfeA-encoded enterobactin receptors of E. coli and Pseudomonas aeruginosa, respectively. Enterobactin prepared from iron-starved E. coli cultures supported growth of B. pertussis and Bordetella bronchiseptica in the presence of the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA). Expression of the bfeA gene was induced by low iron availability, and iron-regulated expression appeared to be dependent upon the presence of the sequence contained within 370 bp upstream of the bfeA structural gene. An internal fragment of the bfeA structural gene and flanking regions were shown by Southern analysis to be highly conserved among Bordetella species. Insertional inactivation of bfeA in both B. pertussis and B. bronchiseptica greatly impaired their ability to grow in the presence of enterobactin and EDDA. These findings suggest that enterobactin produced by other respiratory flora could aid in the colonization of the respiratory tract by Bordetella species.

Amino Acid Sequence↗

PubMed1992

Auxin and growth regulation of rice coleoptile segments : molecular analysis.

Auxin-stimulated elongation of apical segments of rice (Oryza sativa L. cv Arborio) coleoptiles occurring in the first 4 hours of treatment has been studied. Cell extension promoted in the first 2 hours by 10 micromolar indole-3-acetic acid (IAA) is specifically auxin-dependent, whereas after 4 hours, elongation also depends on endogenous production of ethylene. Similar to other systems, rice coleoptile cell elongation stimulated by auxin requires continuous synthesis of RNA and protein. Two-dimensional gel analysis of the in vitro translation products obtained from polyadenylated RNAs extracted from treated and untreated segments after 1 or 4 hours from the initial addition of IAA shows few transcriptional differences. At 60 minutes of treatment, the level of three mRNAs coding for proteins of 22.5, 25, and 33 kilodaltons was moderately enhanced while the disappearance of a 38 kilodalton translation product was observed. Additional repression of another mRNA coding for a 28 kilodalton product begins to show by this time, but becomes more evident after 4 hours treatment. At 4 hours, four IAA-specific mRNA enhancements coding for proteins with molecular masses ranging between 35 to 40 kilodaltons were also observed. We discuss these data in relation to the possible involvement of IAA-mediated transcriptional regulation in growth promotion of rice coleoptiles and, more widely, in control of cell elongation.

Journal Article↗

PubMed1970

Induction and repression of L-arabinose isomerase in bacteriophage-infected Salmonella typhimurium.

The induction of l-arabinose isomerase in Salmonella typhimurium (LT2) is repressed on infection with clear plaque forming mutants (C(1) and C(2)) of the temperate phage P22 (C(+)). However, after infection with C(+) leading to lysogeny, there is a temporary repression. During this period, messenger ribonucleic acid (RNA) for l-arabinose isomerase accumulates. DNA-RNA hybridization data suggest that there is transcription of host DNA during the period of repression. Interference at the level of translation might be responsible for the cessation of induced enzyme synthesis.

Arabinose↗

PubMed2004

Internal loop mutations in the ribosomal protein L30 binding site of the yeast L30 RNA transcript.

Yeast ribosomal protein L30 binds to an asymmetric, purine-rich internal loop in its transcript to repress its own splicing and translation. The protein-bound form of the stem-internal loop-stem RNA is an example of a kink-turn RNA structural motif. Analysis of kink-turn motifs reveals that in (2 + 5) internal loops, the identities of five nucleotides are very important, while the remaining two may be varied. Previous SELEX experiments on the L30 binding site showed an identical pattern of sequence variation with five nucleotides highly conserved and two positions variable. In this work, internal loop residues were mutated and tested for protein binding in vitro and in vivo. The two sheared G-A pairs, which cannot be mutated without severely weakening L30 binding, make sequence specific contacts with other portions of the RNA and L30 protein. In contrast, the lone nucleotide that protrudes into the protein and an unpaired adenosine make no sequence-specific contacts, and may be mutated without compromising L30 binding. The internal loop allows the formation of a very tight bend that brings the two stems together with cross-strand stacking of two adenines and an interhelical ribose contact. Replacement of a ribonucleotide with a deoxynucleotide adjacent to the internal loop weakens protein binding significantly. In the absence of L30, some of the internal loop residues involved in the formation of the kink-turn motif are protected from chemical modification, indicating that some elements of kink-turn structure may form in the free L30 RNA.

Binding Sites↗