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PubMed1995

Temporal regulation of the Xenopus FGF receptor in development: a translation inhibitory element in the 3' untranslated region.

Early frog embryogenesis depends on a maternal pool of mRNA to execute critical intercellular signalling events. FGF receptor-1, which is required for normal development, is stored as a stable, untranslated maternal mRNA transcript in the fully grown immature oocyte, but is translationally activated at meiotic maturation. We have identified a short cis-acting element in the FGF receptor 3' untranslated region that inhibits translation of synthetic mRNA. This inhibitory element is sufficient to inhibit translation of heterologous reporter mRNA in the immature oocyte without changing RNA stability. Deletion of the poly(A) tract or polyadenylation signal sequences does not affect translational inhibition by this element. At meiotic maturation, we observe the reversal of translational repression mediated by the inhibitory element, mimicking that seen with endogenous maternal FGF receptor mRNA at meiosis. In addition, the activation of synthetic transcripts at maturation does not appear to require poly(A) lengthening. We also show that an oocyte cytoplasmic protein specifically binds the 3' inhibitory element, suggesting that translational repression of Xenopus FGF receptor-1 maternal mRNA in the oocytes is mediated by RNA-protein interactions. These data describe a mechanism of translational control that appears to be independent of poly(A) changes.

Animals↗

PubMed1996

Differential regulation of lipoprotein lipase in the macrophage J774.2 cell line by cytokines.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis and in the responses to endotoxin challenge. However, the precise mechanisms by which different cytokines modulate the expression of macrophage LPL activity are poorly understood. The action of six cytokines and bacterial lipopolysaccharide (LPS) on LPL function using the murine J774.2 cell line as a model system has, therefore, been studied. Although exposure to LPS, interleukin 11 (IL-11), tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and IL-1, over the physiological range of concentrations, resulted in a decrease in the heparin-releasable LPL activity, LPL-mRNA levels and LPL-protein content of the cells, stimulation with IL-6 and leukaemia inhibitory factor (LIF) had no effect. The maximum suppression of LPL activity and mRNA levels in the cells by IFN-gamma (60%) was lower than that produced by LPS, IL-11, TNF-alpha and IL-1 (78-97%). Each cytokine displayed a characteristic dose-dependent pattern for the suppression of LPL activity and mRNA levels with IL-11/TNF-alpha being more potent than IFN-gamma/IL-1. More than 80% of the decrease in the LPL activity, at all doses of IL-11, TNF-alpha, IFN-gamma and IL-1, was due to a corresponding reduction in the mRNA levels. The time course of responses to LPS, IL-11, TNF-alpha, IFN-gamma and IL-1 were similar, with the time required to achieve half maximal suppression of LPL activity being between 7 and 9.5 h in each case. These results indicate that LPL in J774.2 macrophages is regulated differentially by various cytokines and that the major control responsible for the reduction of LPL activity by IL-11, TNF-alpha, IFN-gamma and IL-1 is exerted at the level of mRNA metabolism (decreased transcription or RNA stability). The responses identified also displayed several differences to those described previously for adipocytes (e.g. 3T3-L1 cell line), thereby suggesting the existence of potential cell-specific mechanisms for the regulation of LPL by cytokines.

Animals↗

PubMed1990

Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post-transcriptional events.

To shed more light on the controversial findings concerning the functional participation of the highly conserved nut-like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study. We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region. The mutations are located within the genuine rrnB operon cloned on multicopy plasmids. We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry. Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter-proximal 16S compared to the 23S RNA, indicative of a post-transcriptional degradation event. Accordingly, we could demonstrate a reduced amount of free 30S relative to 50S ribosomal subunits in exponentially growing cells. Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor-defective strains. The results show that the leader box A sequence within rRNA operons has important post-transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry. A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus-factor interactions. It is compatible with the previously observed effects of box A in antitermination.

Base Sequence↗

PubMed2005

Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts.

The Sorghum bicolor atp6-1 gene and chimeric atp6 genes with additional maize sequences were introduced into isolated maize mitochondria via electroporation. Transcripts isolated after in vitro incubation of the transformed organelles were then analysed for RNA editing. Transcripts of the S. bicolor atp6-1 gene, and the RNAs obtained from most of chimeric sorghum-maize atp6 gene constructs tested, were not edited. However, the transcript of one engineered chimeric gene comprising the 5'untranslated sequence and a segment of the N-terminal ORF of the maize atp6 combined with the sorghum atp6 core ORF and 3'untranslated sequence was found to be partially edited. We were able to exclude low RNA stability or insufficient editing capacity as the reason for failure to edit in the other instances. Instead, the data indicate that the maize sequence in the edited fusion transcript provides a structural motif or binding site for a transcript-specific editing factor.

Base Sequence↗

PubMed1989

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.

Animals↗

PubMed2001

Operon structure and expression of the genes for benzylsuccinate synthase in Thauera aromatica strain K172.

The first step in anaerobic toluene degradation is the addition of a fumarate cosubstrate to the methyl group of toluene, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme implicated in activating the enzyme by introducing the glycyl radical (BssD). Quantitation of the amounts of benzylsuccinate synthase and activating enzyme showed that both proteins are only synthesized in toluene-grown cells, and that the activating enzyme is present in about 14-fold lower amounts. Two mRNA species of the bss gene cluster were identified, one beginning in front of bssD, and a second in front of bssC. Only the first mRNA 5'-end correlates with a toluene-induced promoter, which is similar to that preceding the bbs operon coding for the further enzymes of toluene catabolism of the same strain. The second mapped 5'-end appears to be generated by endonucleolytic processing. The mRNA segment containing the bssD gene is very short-lived, while that containing the bssCAB genes is more stable. The RNA stability data are consistent with the observed amounts of encoded gene products. Furthermore, the previously known bssDCAB genes are apparently cotranscribed with a fifth gene ( bssE) whose product may function as a putative ATP-dependent chaperone for assembly and/or activation of benzylsuccinate synthase.

Bacterial Proteins↗

PubMed2000

Mutations at three different nuclear loci of Chlamydomonas suppress a defect in chloroplast psbD mRNA accumulation.

In the photosynthetic chloroplast mutant PRB2A of Chlamydomonas reinhardtii the psbD mRNA is unstable. Three strains were isolated, in which the underlying site-directed mutation within the psbD 5' UTR (untranslated region) is suppressed. In all three suppressors, psbD RNA levels and RNA 5' maturation are restored to a varying extent, suggesting a tight coupling of RNA stabilization and 5' processing. Expression of the psbA gene is not compromised in these strains. Genetic crosses revealed that the suppressor mutations affect three unlinked nuclear loci, which may encode new factors involved in psbD gene expression.

Animals↗

PubMed1996

Interleukin-8 production by the human colon epithelial cell line HT-29: modulation by interleukin-13.

1. We have determined which cytokines induce and modulate the production of the chemokine interleukin-8 (IL-8) by the human colonic epithelial cell line HT-29. 2. Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-13 (IL-13), interleukin-10 (IL-10) or vehicle added alone or in combination. The production of IL-8 was determined by enzyme-linked immunosorbent assay (ELISA) and IL-8 messenger RNA expression by Northern blot analysis. 3. The production of IL-8 in unstimulated cells was undetectable by both ELISA and Northern blot analysis. 4. HT-29 cells produced IL-8 following stimulation with IL-1 alpha or TNF-alpha in a time- and a concentration-dependent manner, while IFN-gamma, IL-10 and IL-13 did not induce IL-8 production by HT-29 cells. 5. IL-13 was found to up-regulate significantly (P < 0.01) the IL-1 alpha but not the TNF-alpha-induced IL-8 generation by HT-29 cells. In contrast, IL-10 had no effect on either IL-1 alpha or TNF-alpha-induced IL-8 production. 6. Experiments using cycloheximide demonstrated that this synergistic effect of IL-13 and IL-1 alpha on IL-8 secretion was not through de novo protein synthesis. Using actinomycin-D, we demonstrated that the IL-13 up-regulation was at the level of transcription rather than messenger RNA stability. 7. These findings suggest that colonic epithelial cells have a functional IL-13 receptor, which is coupled to an up-regulation of IL-1 alpha, but not TNF-alpha induced IL-8 generation.

Drug Synergism↗

PubMed2004

The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis.

Ribonucleases have been studied in yeast and bacteria, but their biological significance to multicellular organisms is virtually unknown. However, there is increasing evidence that specific, timed transcript degradation is critical for regulation of many cellular processes, including early development and RNA interference. In this report we have investigated the effects of the 5'-3' exoribonuclease xrn-1 on the development of the nematode worm Caenorhabditis elegans. Silencing of xrn-1 expression using RNA interference results in embryos that fail to complete ventral enclosure, where the outer layer of cells normally closes over the mesoderm in a purse-string movement. Our data suggest that xrn-1 is involved in a critical aspect of epithelial movement and reveal an unexpected link between RNA stability and morphogenesis. Because xrn-1 is highly conserved in all eukaryotes, it is possible that it plays a role in similar morphological processes such as dorsal or thorax closure in Drosophila and wound healing in humans. In contrast to work in human tissue culture cells, where the 3'-5' pathway has been shown to be the most important for degradation of mRNAs, our work shows that the 5'-3' degradation pathway is crucially important at a critical stage of development in C. elegans. We have also investigated whether xrn-1 can influence the response of C. elegans to RNA interference. Our data indicate that xrn-1 plays a facilitating, but not crucial role in this process.

Amino Acid Sequence↗

PubMed1992

Concanavalin A-induced granulocyte-macrophage colony-stimulating factor production in a murine T-cell line is posttranscriptionally controlled.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor (HGF) that regulates the proliferation and differentiation of cells of the myeloid lineage. It can be produced by a variety of cells. One of the major sources of GM-CSF is activated T cells, which transiently produce this HGF. We used the EL-4 thymoma cell line as a model system to address the molecular basis for GM-CSF regulation in T cells. Both concanavalin A (ConA) and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induce GM-CSF expression in EL-4 cells. However, the biological activity of GM-CSF in the supernatants of the TPA-stimulated cells was higher than that of ConA-stimulated cells. To elucidate this difference in biological activity levels, we examined how ConA regulates GM-CSF gene expression in EL-4 cells and compared it to the better-characterized regulation by TPA. Peak mRNA levels of GM-CSF occur 6 h after stimulation with either of these two agents. GM-CSF mRNA levels after ConA treatment are lower and decrease significantly after 10 h compared to TPA treatment, which causes much higher levels that persist for at least 24 h. Neither agent alters GM-CSF gene transcription. Actinomycin D chase experiments show that ConA increases the GM-CSF mRNA half-life from less than 30 to 90 min, whereas TPA prolongs it to greater than 3 h. These results indicate that GM-CSF mRNA induction by ConA (in common with TPA) is regulated predominantly via RNA stabilization and that the difference in prolongation of the mRNA half-life provides the primary explanation for the lower levels of GM-CSF mRNA induced by ConA compared to TPA.

Animals↗

PubMed1990

Light-induced switch in barley psbD-psbC promoter utilization: a novel mechanism regulating chloroplast gene expression.

The synthesis of reaction center protein D2 and mRNAs which encode this protein are differentially maintained at high levels in mature barley chloroplasts. To understand the differential maintenance of psbD mRNA abundance, we have studied the transcription and the RNAs produced from the psbD-psbC operon in plastids of light and dark-grown barley seedlings. Ten psbD-psbC RNAs synthesized in dark-grown barley share four different 5'-ends, two of which arise by transcription initiation, and one of which is generated by 5'-processing of longer psbD-psbC transcripts. Illumination of dark-grown barley causes the decline of these ten transcripts, and the accumulation of two different psbD-psbC RNAs. Capping assays, in vitro transcription and RNA processing experiments and treatment of plants with tagetitoxin (a selective inhibitor of chloroplast transcription), indicate that the light-induced transcripts arise by transcription initiation. Run-on transcription and RNA quantitation experiments provide evidence that both light-induced transcription and RNA stability play roles in the accumulation of the light-induced RNAs. These data document a novel mechanism for regulating plastid gene expression involving a light-induced switch in psbD-psbC promoter utilization.

Base Sequence↗

PubMed1995

AU-rich elements: characterization and importance in mRNA degradation.

Adenylate/uridylate-rich elements (AREs) are found in the 3' untranslated region (UTR) of many messenger RNAs (mRNAs) that code for proto-oncogenes, nuclear transcription factors and cytokines. They represent the most common determinant of RNA stability in mammalian cells. Moreover, ARE-directed mRNA degradation is influenced by many exogenous factors, including phorbol esters, calcium ionophores, cytokines and transcription inhibitors. These observations suggest that AREs play a critical role in the regulation of gene expression during cell growth and differentiation, and in the immune response.

Adenine↗

PubMed2001

Regulation of aromatase by nuclear receptors.

We investigated the effects of a nuclear receptor system constituted by retinoid X receptor (RXR) and its heterodimer partner on the aromatase activity in a cultured MCF-7 human breast cancer cell line and also in human ovarian granulosa cells, using each selective ligand for retinoic acid receptor, RAR (TTNPB), retinoid X receptor, RXR (LG100268), PPARgamma (troglitazone), and vitamin D3 receptor (cholecalciferol). In MCF-7 cells, the combined treatment with TTNPB and LG100268 caused a dramatic stimulation of the aromatase activity. The combined treatment with other ligand and LG100268 had little or no effect on the aromatase activity. The increase in the aromatase activity by TTNPB plus LG100268 was accompanied by an increase in the P450arom mRNA levels, which was also found to be related to the specific usage of promoter 1a of the CYP19 gene. These results suggest that a nuclear receptor system constituted by a RAR:RXR heterodimer is involved in the regulation of aromatase activity in MCF-7 breast cancer cells. In cultured human ovarian granulosa cells obtained from patients who underwent in vitro fertilization, troglitazone or LG100268 alone decreased the aromatase activity, while the combined treatment caused an even greater reduction in this activity. Little effect of other specific ligands for RXR heterodimer partners may support the notion that the effects of troglitazone and/or LG100268 in human granulosa cells may be mediated through the specific activation of PPARgamma:RXR heterodimer system. Since similar manners of effects of several PPARgamma ligands and/or LG100268 on the aromatase activity were observed in a newly established human ovarian granulosa cancer cell line, KGN, we performed the detailed analysis of the mechanisms of these effects using this cell line. As a result, the inhibitory effect of aromatase activity by troglitazone plus LG100268 was accompanied by the decrease of P450arom mRNA level. Furthermore, the loss of P450arom expression was considered to be due to both the decreased transcription and rapid degradation of its RNA based on the studies of nuclear run-on assay and RNA stability assay. In conclusion, RAR:RXR and PPARgamma:RXR heterodimer nuclear receptor systems may be other important modulators of estrogen production in human breast cancer cells and ovarian granulosa cells, respectively.

Aromatase↗

PubMed2000

RNA-protein interactions in the control of stability and localization of messenger RNA (review).

Growing evidence demonstrates the importance of regulating mRNA localization, stability and translation, in the control of gene expression, both in development and in differentiated cells. The signals responsible for specific regulation of mRNA metabolism reside in the RNA message itself: both 5' and 3' to the coding region, all transcripts contain variable lengths of untranslated sequences (5'-UTR and 3'-UTR) which contain the binding sites for a number of RNA-binding proteins (RBPs). Most RBPs assemble on the message at the moment of transcription, thus determining the future fate of the transcript from the very beginning. We discuss possible mechanisms through which mRNA, leaving from the nucleus as an RNA-protein complex, might reach its final intracellular destinations and how its access to the translational apparatus might be regulated in time and space. We also focus on a few known examples of aberrant RNA-protein interactions associated with human diseases, including cancer.

3' Untranslated Regions↗

PubMed1993

Estrogen regulation of proto-oncogenes coding for nuclear proteins.

Estrogen hormones are known to exert a complex influence on development and function of the female reproductive organs of vertebrates by regulating cell growth and differentiation, as well as to be implicated in oncogenesis and maintenance of tumor growth. Estrogen acts on cells via interaction with an intracellular receptor, which, like all receptors for steroid hormones, is a trans-acting transcription enhancer factor activated by the cognate ligand and capable of binding to specific, cis-acting enhancer elements usually located within the 5'-flanking regions of target genes. Additionally, estrogen regulates gene expression by influencing mRNA stability or via interaction of the estrogen receptor with transcription regulatory factors. This article reviews data indicating that estrogen directly activates (primary activation) expression of proto-oncogenes codifying for nuclear proteins that, in turn, are responsible for indirect (secondary) activation of other genes. This cascade mechanism of gene activation is likely to progress for several more steps and allows us to envisage how estrogen can direct a complex task such as cell reproduction. Among proto-oncogenes codifying for nuclear proteins, we focus on fos, jun, myc, and related genes. The mechanisms of regulation of these genes by estrogen, including regulation of transcription, messenger RNA stabilization, and protein-protein interaction, are reviewed.

Animals↗

PubMed1993

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.

Base Composition↗

PubMed1995

Mutations in the adenovirus-encoded single-stranded DNA binding protein that result in altered accumulation of early and late viral RNAs.

Adenovirus encodes a 72-kDa single-stranded DNA binding protein (DBP) that is necessary for viral DNA replication and is involved in controlling viral RNA metabolism. Studies of temperature-sensitive (ts) and site-directed mutants of the DBP have identified at least four regions of the protein involved in binding to single-stranded DNA and hence in DNA replication. Two of the ts mutants, Ad2ts111A and Ad2ND1+ts23, are deficient in DNA binding and in supporting in vitro DNA replication. Their effects on viral RNA metabolism are presented here. At early and late times of infection, accumulation of RNAs from the viral E3 region is increased up to sevenfold in Ad2ts111A- and Ad2ND1+ts23-infected cells relative to wild-type virus. This effect is temperature-independent and seems to involve nuclear RNA stability. Steady-state levels of the viral E1B and E4 RNAs increase following the onset of viral DNA replication in cells infected by the wild-type virus, but not in cells infected by ts111A or ts23. The increase in E1B and E4 RNAs at late times of infection is due to a stabilization of the mRNA. The steady-state levels of L3 and L5 RNAs are two- to fourfold higher in cells infected with ts23 and ts111A than with wild-type virus. None of these differences were observed following infection of cells with a temperature-independent revertant of ts111, indicating that the mutations in the DBP were responsible for the phenotypes. However, for the E3 effect, the change brought about by the mutations in the DBP does not seem to be the result of a change in the normal function of the protein, as an essentially DBP-negative virus (Ad5d/802) shows no differences in E3 RNA metabolism compared to wild-type virus at early times of infection. These results demonstrate that mutation of amino acids 280 and 282 of the DBP significantly perturbs the normal regulation of viral RNA metabolism. These effects clearly differ from the phenotypes observed with adenoviruses containing mutations in other early genes and from those ascribed to the ts125 mutation by others. These results are discussed in terms of the mechanisms by which early and late viral RNA metabolism are controlled and the possible effects of the DBP mutations on them.

Adenoviruses, Human↗

PubMed1999

Transcripts containing the 5' untranslated regions of the plastid genes psbA and psbB from higher plants are unstable in Chlamydomonas reinhardtii chloroplasts.

The 5' regions of chloroplast genes contain cis-acting regulatory elements including promoters, and determinants of RNA stability and translation. In this work I examined whether the 5' regions of the spinach psbB and the wheat psbA genes can drive the expression of an aadA reporter gene in chloroplasts of the unicellar green alga Chlamydomonas reinhardtii. Both plant 5' sequences confer aadA-dependent, spectinomycin resistance on Escherichia coli, but not on the alga following integration into its chloroplast genome. Northern and run-on transcription analyses reveal that the plant promotors are active in C. reinhardtii but that the resulting chimeric transcripts are unstable. Therefore, the data suggest differences between higher plants and green algae with respect to the molecular mechanisms underlying plastid RNA metabolism.

5' Untranslated Regions↗