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Results for “Translational repression”

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PubMed2004

Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva.

In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebrafish embryos. Like the human CYP3A genes, CYP3A65 transcription in the foregut region was enhanced by treatment of the zebrafish larvae with the steroid dexamethasone and the macrocyclic antibiotic rifampicin. Differing from mammalian CYP3A genes, CYP3A65 transcription was also elicited by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) during early larval stages. Repression of AHR2 translation by antisense morpholino oligonucleotides abrogated both of constitutive and TCDD-stimulated CYP3A65 transcription in larval intestine. These findings suggested that the AHR2 signaling pathway plays an essential role in CYP3A65 transcription.

Animals↗

PubMed2006

Structural and thermodynamic basis for the enhanced transcriptional control by the human papillomavirus strain-16 E2 protein.

Strain 16 of the human papillomavirus is responsible for the largest number of cases of cervical cancers linked to this virus, and the E2 protein is the transcriptional regulator of all viral genes. We present the first structure for the DNA binding domain of HPV16 E2 bound to DNA, and in particular, to a natural cognate sequence. The NMR structure of the protein backbone reveals that the overall conformation remains virtually unchanged, and chemical shift analysis of the protein bound to a shorter DNA duplex uncovered a contact out of the minimal E2 DNA binding site, made by lysine 349. This contact was confirmed by titration calorimetry and mutagenesis, with a contribution of 1.0 kcal mol(-)(1) to binding energy. HPV16 E2 has the highest DNA binding affinity and exerts a strict transcriptional control, translated into the repression of the E6 and E7 oncogenes. These novel features provide the structural and thermodynamic basis for this tight transcriptional control, the loss of which correlates with carcinogenesis.

Amino Acid Sequence↗

PubMed1994

Crystal structure of an RNA bacteriophage coat protein-operator complex.

The RNA bacteriophage MS2 is a convenient model system for the study of protein-RNA interactions. The MS2 coat protein achieves control of two distinct processes--sequence-specific RNA encapsidation and repression of replicase translation--by binding to an RNA stem-loop structure of 19 nucleotides containing the initiation codon of the replicase gene. The binding of a coat protein dimer to this hairpin shuts off synthesis of the viral replicase, switching the viral replication cycle to virion assembly rather than continued replication. The operator fragment alone can trigger self-assembly of the phage capsid at low protein concentrations and a complex of about 90 RNA operator fragments per protein capsid has been described. We report here the crystal structure at 3.0 A resolution of a complex between recombinant MS2 capsids and the 19-nucleotide RNA fragment. It is the first example of a structure at this resolution for a sequence-specific protein-RNA complex apart from the transfer RNA synthetase complexes. The structure shows sequence-specific interactions between conserved residues on the protein and RNA bases essential for binding.

Base Sequence↗

PubMed2006

Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae.

Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR). How the UPR is downregulated is not well understood. Inositol requirement 1 (Ire1) is an endoplasmic reticulum transmembrane UPR sensor in Saccharomyces cerevisiae. When the UPR is triggered, Ire1 is autophosphorylated, on Ser 840 and Ser 841, inducing the cytosolic endonuclease activity of Ire1, thereby initiating the splicing and translational de-repression of HAC1 mRNA. Homologous to Atf/Creb1 (Hac1) activates UPR transcription. Here, we report that the dose-dependent cell-cycle regulator 2 (Dcr2) phosphatase functionally and physically interacts with Ire1. We identified genetic interactions between DCR2 and genes, including IRE1, which are involved in secretory processes. Overexpression of DCR2, but not of a catalytically inactive DCR2 allele, significantly delays HAC1 splicing and sensitizes cells to the UPR. Furthermore, Dcr2 physically interacts in vivo with Ire1-S840E,S841E, which mimics phosphorylated Ire1, and Dcr2 de-phosphorylates Ire1 in vitro. Our results are consistent with de-phosphorylation of Ire1 being a mechanism for antagonizing UPR signalling.

Animals↗

PubMed2001

Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation.

When Escherichia coli cells are shifted to low temperatures (e.g. 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome. At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C. However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.

Adaptation, Biological↗

PubMed1972

Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17.

The binding of a few molecules [1-6] of RNA bacteriophage coat protein to 1 molecule of RNA represses in vitro translation of the RNA synthetase cistron. Digestion of the complex, R17 coat protein-R17 RNA, by T1 RNase yields an RNA fragment bound to the coat protein. The nucleotide sequence of this fragment (59 residues) reveals that it contains the punctuation signal between the coat protein and RNA synthetase cistrons, suggesting that this is the site on the RNA where the coat protein acts as a translational repressor.

Acid Phosphatase↗

PubMed2000

Circadian clock-protein expression in cyanobacteria: rhythms and phase setting.

The cyanobacterial gene cluster kaiABC encodes three essential circadian clock proteins: KaiA, KaiB and KaiC. The KaiB and KaiC protein levels are robustly rhythmical, whereas the KaiA protein abundance undergoes little if any circadian oscillation in constant light. The level of the KaiC protein is crucial for correct functioning of the clock because induction of the protein at phases when the protein level is normally low elicits phase resetting. Titration of the effects of the inducer upon phase resetting versus KaiC level shows a direct correlation between induction of the KaiC protein within the physiological range and significant phase shifting. The protein synthesis inhibitor chloramphenicol prevents the induction of KaiC and blocks phase shifting. When the metabolism is repressed by either translational inhibition or constant darkness, the rhythm of KaiC abundance persists; therefore, clock protein expression has a preferred status under a variety of conditions. These data indicate that rhythmic expression of KaiC appears to be a crucial component of clock precession in cyanobacteria.

Bacterial Proteins↗

PubMed2004

The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development.

MicroRNAs (miRNAs) are endogenous 21-24-nt RNAs that can down-regulate gene expression by pairing to the messages of protein-coding genes to specify mRNA cleavage or repression of productive translation. They act within the RNA-induced silencing complex (RISC), which in animals contains a member of the Argonaute family of proteins. In the present study, we show that Arabidopsis ago1 mutants have increased accumulation of mRNAs known to be targeted for cleavage by miRNAs. In hypomorphic ago1 alleles, this compromised miRNA function occurs without a substantial change in miRNA accumulation, whereas in null alleles it is accompanied by a drop in some of the miRNAs. Therefore, AGO1 acts within the Arabidopsis miRNA pathway, probably within the miRNA-programmed RISC, such that the absence of AGO1 destabilizes some of the miRNAs. We also show that targeting of AGO1 mRNA by miR168 is needed for proper plant development, illustrating the importance of feedback control by this miRNA. Transgenic plants expressing a mutant AGO1 mRNA with decreased complementarity to miR168 overaccumulate AGO1 mRNA and exhibit developmental defects partially overlapping with those of dcl1, hen1, and hyl1 mutants showing a decrease in miRNA accumulation. miRNA targets overaccumulate in miR168-resistant plants, suggesting that a large excess of AGO1 protein interferes with the function of RISC or sequesters miRNAs or other RISC components. Developmental defects induced by a miR168-resistant AGO1 mRNA can be rescued by a compensatory miRNA that is complementary to the mutant AGO1 mRNA, proving the regulatory relationship between miR168 and its target and opening the way for engineering artificial miRNAs in plants.

Amino Acid Sequence↗

PubMed1996

Translational control of poly(A)-binding protein expression.

Poly(A)-binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract. In many eukaryotic cells, this mRNA is inefficiently translated. The 5' untranslated region (UTR) of PABP mRNA has several adenine-rich regions which may serve as the PABP-binding sites to control its translation by a feed-back mechanism. This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell-free system. Results of our studies show that removal of the putative PABP-binding sites from the 5' UTR of this mRNA enhances its translation in the rabbit reticulocyte cell-free system. Furthermore, in vitro translation of the full-length PABP mRNA was inhibited by addition of purified PABP to the cell-free system. In contrast, translation of truncated mRNA lacking the putative PABP-binding sites at the 5' UTR was not inhibited by exogenous PABP. We have also tested the ability of purified PABP to bind to the 5' UTR of PABP mRNA using ultraviolet-mediated covalent cross-linking of RNA and proteins in vitro. Our results show that exogenous PABP binds to the 5' UTR of its full-length mRNA. Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5' UTR. The adenine-rich regions are located within this segment of PABP mRNA. Following incubation of PABP mRNA in the reticulocyte lysate cell-free system under conditions of mRNA translation, the polysomal and non-translated free mRNA fractions were separated by centrifugation. Analysis of free and polysomal mRNA-protein (mRNP) complexes following ultraviolet-induced cross-linking showed that the free mRNP population was preferentially enriched in PABP. Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed-back mechanism.

DNA, Complementary↗

PubMed1998

Controlling gene expression in living cells through small molecule-RNA interactions.

Short RNA aptamers that specifically bind to a wide variety of ligands in vitro can be isolated from randomized pools of RNA. Here it is shown that small molecule aptamers also bound their ligand in vivo, enabling development of a method for controlling gene expression in living cells. Insertion of a small molecule aptamer into the 5' untranslated region of a messenger RNA allowed its translation to be repressible by ligand addition in vitro as well as in mammalian cells. The ability of small molecules to control expression of specific genes could facilitate studies in many areas of biology and medicine.

Animals↗

PubMed2001

Abundant early expression of gpUL4 from a human cytomegalovirus mutant lacking a repressive upstream open reading frame.

The human cytomegalovirus UL4 gene encodes a 48-kDa glycoprotein, expression of which is repressed at the translational level by a short upstream open reading frame (uORF2) within the UL4 transcript leader. Mutation of the uORF2 initiation codon in the viral genome eliminates ribosomal stalling at the uORF2 termination site, resulting in early and abundant gpUL4 protein synthesis. This mutation does not appear to affect viral replication kinetics in human fibroblasts. These results reveal that the unusual uORF2 inhibitory mechanism is a principal determinant of the abundance and timing of gpUL4 expression but is nonessential for replication in cell culture.

Cell Line↗

PubMed2026

Riboregulation: a non-canonical tau function.

Almost since its discovery, tau protein has perplexed scientists and clinicians with its varied roles in physiology as well as its appearance as phosphorylated protein aggregates of various structures in many neurodegenerative diseases. Tau plays a role in microtubule stabilization, but from the earliest of studies, tau has also been observed to bind to RNA, with recent research suggesting tau has a higher affinity for some RNA species compared to microtubules. In the context of disease, tau dysfunction potentiates disruptions to RNA metabolism, including the perturbation of mRNA splicing, impairment of translation, de-repression of transposable elements, and alteration of RNA export and degradation. Tau aggregates directly sequester diverse RNA species and RNA binding proteins. Emerging evidence reinforces the characterization of tau as an RNA binding protein, highlighting questions about both the physiological and disease-related functions of this direct RNA binding. The disparate structure of tau in normal and various disease states makes teasing apart the various impacts on RNA and regulation a more difficult puzzle requiring future study. In this review, we summarize the evidence for tau's role in RNA biology, including as an RNA binding protein.

tau Proteins↗

PubMed2002

Manipulation of the cell cycle by human cytomegalovirus.

The human cytomegalovirus-induced changes to the transcriptome and proteome of infected cells in many ways resemble an abortive mitogenic response. The virus induces quiescent cells to re-enter the cell cycle, but they are prevented from entering the S phase, where the synthesis of the cellular genome would compete with that of the virus for the available precursors for DNA replication. The mechanisms of these cell cycle alterations include transcriptional induction and repression, post-translational modifications and changes in protein stability. Essentially every class of cell cycle regulators is affected, and some of the key proteins are targeted by multiple different mechanisms. While the effects on cell cycle progression of viral infection, and of individual viral genes outside the context of viral infection have been described, it is now important to synthesize these two experimental approaches to gain a more complete understanding of how and why human cytomegalovirus infection affects cell cycle progression.

Animals↗

PubMed2006

Is the anti-sarcoma and anti-viral cytokine "plasma factor" a novel chicken Y-box protein?

A line of research beginning in the early 1960s with the observation that West Nile virus and, later, several strains of rabies virus could inhibit the development of the Rous sarcoma virus-induced tumor in the wing-web of chicken (a "sarcoma-blockade") eventually culminated in the characterization of a 14-kDa circulating anti-sarcoma and anti-viral activity christened "plasma factor" (PF) which, unlike the interferons, inhibited the replication of diverse RNA-containing viruses, but not of any DNA-containing viruses. The possibility that this 14 kDa protein represented a novel antiviral cytokine has been strengthened by analysis of partial amino acid sequencing data which suggest that this 14-kDa cytokine may correspond to the 127-amino acid-long chicken YB2-like protein (Locus: XP_423576) deduced very recently from the genomic sequencing of chicken. Biologically, proteins of the Y-box family (such as chicken YB1 and YB2) not only bind DNA and thus regulate transcription but also bind single-stranded RNA in a sequence-specific and reversible manner, repress viral RNA translation, inhibit retroviral transformation of chicken fibroblasts, and are known to regulate transcription of human immunodeficiency virus and hepatitis B virus. Taken together, the available data point to a novel anti-viral cytokine with a novel mechanism of action.

Amino Acid Sequence↗

PubMed1989

Regulation of endothelial cell coagulant properties. Modulation of tissue factor, plasminogen activator inhibitors, and thrombomodulin by phorbol 12-myristate 13-acetate and tumor necrosis factor.

The procoagulant response of endothelium to many stimuli alters the expression of tissue factor, thrombomodulin, and plasminogen activator inhibitors (PAI) PAI-1 and PAI-2. The regulation of these proteins was examined in cultured human endothelial cells treated with phorbol myristate acetate (PMA) or tumor necrosis factor (TNF). Unstimulated cells contained approximately 670 PAI-1 and approximately 100 thrombomodulin mRNA molecules/cell, whereas tissue factor and PAI-2 mRNAs were not detectable. By 3-5 h, PMA or TNF induced both tissue factor and PAI-2 to approximately 150-420 mRNA molecules/cell and both mRNAs declined to basal levels within several hours; however, PAI-1 and thrombomodulin mRNA levels did not change. Nuclear runoff assays showed that PMA, TNF, or cycloheximide induced transcription of the tissue factor gene, whereas the genes for thrombomodulin, PAI-1, and PAI-2 apparently were transcribed at the same relative rate in the presence or absence of these agents. Treatment of cells with cycloheximide stabilized tissue factor and PAI-2 mRNAs and increased their induction by PMA or TNF. The synthesis of tissue factor, PAI-1, and PAI-2 proteins paralleled their mRNA levels. The effects of TNF were similar to those of PMA with one exception. In contrast to PMA, TNF reduced thrombomodulin activity approximately 80% with no change in thrombomodulin mRNA levels. Thus, PAI-2 may be induced by inhibiting mRNA degradation. Tissue factor can be induced by stimulating transcription and potentially by inhibiting mRNA degradation. Thrombomodulin can be repressed by a translational or posttranslational mechanism. PAI-1 was not regulated under the conditions studied. The different effects of PMA and TNF on thrombomodulin expression indicate that some effects of TNF are not mediated solely by protein kinase C.

Cell Line↗

PubMed1980

[Role of protein synthesis in the process of degradation of anomalous proteins in Escherichia coli cells].

The degradation of three types of anomalous proteins, e. g. those containing the arginine analog kanavanine; polypeptides synthesized in the presence of puromycin (100 micrograms/ml) under slight inhibition of total translation, and polypeptides synthesized under amino acid deficiency, was studied. In order to measure the rate of proteolysis, the E. coli cells were labelled for 5 min with [14C]-phenylalanine and then transferred to a complete medium. The loss of TCA-insoluble material was taken as a measure of proteolysis. While the normal total protein of E. coli cells was degraded at the rate of 2--8% per hour, the canavanine-containing proteins were degraded at the rate of 30--40% per hour. The polypeptides synthesized in the presence of puromycin were degraded at the rate of 10--15% per hour, while the polypeptides formed under amino acid starvation--at the rate of 7--8% per hour. The rate of proteolysis of canavanine-containing polypeptides was two times lower under inhibition of translation by chloramphenicol, tetracycline of kasugamicin, while the rate of degradation of two other types of anomalous polypeptides was significantly increased. Tetracycline at concentrations significantly exceeding those sufficient for maximal inhibition of translation, practically completely repressed the proteolysis of canavanine-containing proteins. No such tetracycline activity was observed in the presence of 20 mM Mg2+, which was assumed to be dependent on the complexon-forming ability of the antibiotic.

Amino Acids↗

PubMed2001

Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo.

Development of the Drosophila abdomen requires repression of maternal hunchback (hb) mRNA translation in the posterior of the embryo. This regulation involves at least four components: nanos response elements within the hb 3' untranslated region and the activities of Pumilio (PUM), Nanos (NOS), and Brain tumor. To study this regulation, we have developed an RNA injection assay that faithfully recapitulates the regulation of the endogenous hb message. Previous studies have suggested that NOS and PUM can regulate translation by directing poly(A) removal. We have found that RNAs that lack a poly(A) tail and cannot be polyadenylated and RNAs that contain translational activating sequences in place of the poly(A) tail are still repressed in the posterior. These data demonstrate that the poly(A) tail is not required for regulation and suggest that NOS and PUM can regulate hb translation by two mechanisms: removal of the poly(A) tail and a poly(A)-independent pathway that directly affects translation.

3' Untranslated Regions↗

PubMed2000

Tumor necrosis factor-alpha mRNA remains unstable and hypoadenylated upon stimulation of macrophages by lipopolysaccharides.

TNF-alpha gene expression is regulated at transcriptional and post-transcriptional levels in mouse macrophages. The post-transcriptional regulation is mediated by the AU-rich element (ARE) located in the TNF-alpha mRNA 3' untranslated region (UTR), which controls its translation and stability. In resting macrophages, the ARE represses TNF-alpha mRNA translation. Activation of macrophages with various agents [for example lipopolysaccharide (LPS), viruses] results in translational derepression, leading to the production of high levels of TNF-alpha. TNF-alpha ARE has also been shown to confer mRNA instability as its deletion from the mouse genome leads to an increase in the TNF-alpha mRNA half-life [Kontoyiannis, D., Pasparakis, M., Pizzaro, T., Cominelli, F. & Kollias, G. (1999) Immunity 10, 387-398]. In this study, we measured the half-life as well as the poly(A) tail length of TNF-alpha mRNA in the course of macrophage activation by LPS. We report that TNF-alpha mRNA is short lived even in conditions of maximal TNF-alpha synthesis. Moreover, TNF-alpha mRNA is hypoadenylated in a constitutive manner. These results reveal that TNF-alpha mRNA rapid turnover does not constitute a regulatory step of TNF-alpha biosynthesis in macrophages and that TNF-alpha mRNA translational activation upon LPS stimulation is not accompanied by a change of poly(A) tail length.

3' Untranslated Regions↗