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At least 1,207 records · Page 67Linked to original sources
PubMed1988

Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro.

An extract of whole oocytes (oocyte S150) differentially transcribes Xenopus oocyte and somatic 5S RNA genes. In the oocyte S150, transcription complexes with different stabilities are assembled onto oocyte and somatic 5S DNA. The stability of the transcription complex is dependent on activities present in a fraction containing transcription factor TFIIIC. This fraction stabilizes the binding of the positive transcription factor TFIIIA to a somatic 5S RNA gene much more efficiently than it does to an oocyte gene. The oocyte S150 transcription extract is deficient in TFIIIC such that supplementation with a fraction enriched in this transcription factor selectively stimulates oocyte 5S DNA transcription. Previously it has been shown that an egg extract deficient in TFIIIA selectively transcribes somatic 5S RNA genes. Thus under conditions where there is differential stability of transcription complexes, limitation of either TFIIIA or TFIIIC may exaggerate the differential expression of two genes.

Animals↗
PubMed2006

Small dsRNAs induce transcriptional activation in human cells.

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21(WAF1/CIP1) (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5' end of the antisense strand, or "seed" sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.

Argonaute Proteins↗
PubMed1991

Regulation of collagen gene expression by transformed human fibroblasts: decreased type I and type III collagen RNA transcription.

The regulation of collagen gene expression in normal diploid human fetal fibroblasts (KMS-6 cells), and fibroblasts immortally transformed by treatment of KMS-6 with Co-60 gamma rays (KMST-6 cells) was compared to that of ones tumorigenically transformed by treatment of KMST-6 cells with Harvey murine sarcoma virus (KMST-6-Ras cells). Synthesized collagenous protein decreased to approximately 30% of that of normal fetal fibroblasts in both transformed cell lines, and the relative rate of collagen synthesis to total protein synthesis decreased about sixfold in KMST-6 cells and twelvefold in KMST-6-Ras cells. The m-RNA levels of type I collagen in both of these cell lines decreased to approximately 20% of that of the control fibroblasts, whereas type III collagen m-RNA levels decreased to only 9% of that of the control. The copy number of the collagen gene in both transformed cell lines was unaltered. The transcriptional rates of collagen alpha 1(I) and collagen alpha 1(III) in both cell lines decreased to 20% and 7% respectively of that of control. These data indicate that collagen synthesis was reduced at the transcriptional level in these transformed human fibroblasts.

Cell Line, Transformed↗
PubMed2006

Messenger RNA turnover and its regulation in herpesviral infection.

The ability to regulate cellular gene expression is a key aspect of the lifecycles of a diverse array of viruses. In fact, viral infection often results in a global shutoff of host cellular gene expression; such inhibition serves not only to ensure maximal viral gene expression without competition from the host for essential machinery and substrates but also aids in evasion of immune responses detrimental to successful viral replication and dissemination. Within the herpesvirus family, host shutoff is a prominent feature of both the alpha- and gamma-herpesviruses. Intriguingly, while both classes of herpesviruses block cellular gene expression by inducing decay of messenger RNAs, the viral factors responsible for this phenotype as well as the mechanisms by which it is achieved are quite distinct. However, data suggest that the host shutoff functions of alpha- and gamma-herpesviruses are likely achieved both through the activity of virally encoded nucleases as well as via modulation of cellular RNA degradation pathways. This review highlights the processes governing normal cellular messenger RNA decay and then details the mechanisms by which herpesviruses promote accelerated RNA turnover. Parallels between the viral and cellular degradation systems as well as the known interactions between viral host shutoff factors and the cellular RNA turnover machinery are highlighted.

Animals↗
PubMed1996

Chloroplast mRNA 3'-end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins.

In the absence of efficient transcription termination correct 3'-end processing is an essential step in the synthesis of stable chloroplast mRNAs in higher plants. We show here that 3'-end processing in vitro involves endonucleolytic cleavage downstream from the mature terminus, followed by exonucleolytic processing to a stem-loop within the 3'-untranslated region. These processing steps require a high molecular weight complex that contains both endoribonucleases and an exoribonuclease. In the presence of ancillary RNA binding proteins the complex correctly processes the 3'-end of precursor RNA. In the absence of these ancillary proteins 3'-end maturation is prevented and plastid mRNAs are degraded. Based on these results we propose a novel mechanism for the regulation of mRNA 3'-end processing and stability in chloroplasts.

Amino Acid Sequence↗
PubMed2005

Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins.

The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3'-untranslated region (3'-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3'-UTR. This site resembles ELAV-binding sites identified previously in heterologous targets, both for its nucleotide sequence and its significant affinity for ELAV (K(d) 40 nM). This finding supports our model that elav autoregulation depends upon direct interaction between ELAV and elav RNA. We narrowed down the best binding site to a 20 nt long sequence A(U5)A(U3)G(U2)A(U6) in an alternative 3' exon. We propose and test a model in which the regulated use of this alternative 3' exon is involved in normal elav regulation. Found in NEurons (FNE), another neuronal RNA-binding protein paralogous to ELAV, also binds this site. These observations provide a molecular basis for the in vivo interactions reported previously between elav and fne.

3' Untranslated Regions↗
PubMed1990

Translational regulation by HIV leader RNA, TAT, and interferon-inducible enzymes.

Every step in the replication cycle of HIV provides unique opportunities for controlling the progression of AIDS. In this regard, virus protein synthesis should be an important target for limiting HIV multiplication in cells. Molecular mechanisms in the regulation of HIV protein synthesis were therefore investigated in the context of interferon action. The interferon-inducible enzymes, 2-5A synthetase and dsRNA-dependent protein kinase, which can inhibit translation were activated by HIV-1 leader RNA. In cell-free systems, leader RNA and Tat protein of HIV inhibited and enhanced translation, respectively. An intriguing interplay of these viral and host factors were shown to influence the rate of translation in vitro. A model describing opposing actions of HIV Tat protein and interferon in HIV replication is represented.

2',5'-Oligoadenylate Synthetase↗
PubMed2004

AU-rich elements and the control of gene expression through regulated mRNA stability.

The regulation of gene expression is a fundamental cellular process that is controlled at multiple levels. Abnormal regulation of gene expression has been directly implicated in the pathogenesis of some diseases of animals and humans and may contribute to the disease process in unrecognized ways in many others. Furthermore, novel treatment strategies for a number of different diseases may hinge upon our ability to exploit mechanisms that normally alter the expression of endogenous genes. While the study of gene regulation has traditionally focused on transcription as a major regulator of gene expression, it has recently become apparent that the post-transcriptional control of gene expression may play an equally important role. In particular, rapid, context-specific regulation of the stability of mRNA transcripts encoding highly active proteins, such as cytokines, growth factors, oncogenes and cell-cycle regulators, appears to play a key role in the control of these molecules and the processes they mediate. Many of the known regulatory pathways for mRNA stability involve proteins that interact with specific AU-rich elements in the 3'-untranslated region of the transcript. This review will address some important aspects of the normal regulation of mRNA stability and known or potential contributions of RNA stability regulation to health and disease.

Animals↗
PubMed1989

Regulation of extracellular matrix RNA levels in cultured smooth muscle cells. Relationship to cellular quiescence.

We have examined how the alpha 2(I), alpha 1(III), alpha 1(IV), and alpha 2(V) collagen genes as well as the thrombospondin and fibronectin genes are regulated by conditions that modulate the proliferation of smooth muscle cells in vitro. We find that smooth muscle cells (SMC) arrested by low serum containing media continue to express similar or slightly lower levels of mRNA for all the genes studied except type III collagen which exhibited a 5-fold increase. By contrast, when SMC are density-arrested, a coordinate increase in mRNA levels for all the extracellular matrix genes studied is observed. The increases range from approximately 1.5-fold for fibronectin to 5-fold for type III collagen. The mRNA levels for beta-tubulin and vimentin, however, are decreased in these density-arrested cells. Interestingly, the time course of increase for type IV collagen and fibronectin can be distinguished from the other extracellular matrix genes and best correlates with an increase in cell-cell contact. The increase in steady-state RNA levels for alpha 1(IV) collagen is not shared by fibroblasts similarly treated and may provide a useful marker for SMC in culture. Heparin, a potent inhibitor of SMC growth, increased the level of type III collagen mRNA about 2.5-fold and also increased the level of alpha 2(V) collagen and fibronectin. Transforming growth factor-beta is found to be a cell density-dependent modulator of SMC growth. Surprisingly, transforming growth factor-beta had little effect on most of the extracellular matrix genes studied. However, transforming growth factor-beta treatment of SMC increased thrombospondin mRNA levels about 4-fold, whereas type IV collagen mRNA exhibited a 2-fold increase. We conclude that type III collagen mRNA levels are positively and tightly coupled to SMC quiescence. The effect of cellular quiescence on other extracellular matrix mRNAs is complex but, in general, positive. This is particularly evident in SMC arrested by high cell density.

Animals↗
PubMed1985

[Effect of a serum inhibitor on hematopoiesis regulation: in vitro effect on RNA synthesis in bone marrows of rats].

Hematopoiesis is regulated by several hormones and protein factors, among them, erytropoietin (Ep) and granulopoietin (CSF: Colony Stimulating Factor) play the fundamental control activities inducing the differentiation of stem cells to red and white cells series, respectively. The action of serum inhibitors and cyclic nucleotides on hematopoietic process is not well understood at present time. In this work a rat serum-inhibiting activity was detected that diminished the in vitro [3H] uridine incorporation into rat bone marrow whole RNA. The experiments show that the inhibitory may have a platelet origin.

Animals↗
PubMed2002

Dendritic BC1 RNA: functional role in regulation of translation initiation.

In neurons, local protein synthesis in synaptodendritic microdomains has been implicated in the growth and plasticity of synapses. Prerequisites for local translation are the targeted transport of RNAs to distal sites of synthesis in dendrites and translational control mechanisms to limit synthesis to times of demand. Here we identify dendritic BC1 RNA as a specific repressor of translation. Experimental use of internal ribosome entry mechanisms and sucrose density gradient centrifugation showed that BC1-mediated repression targets translation at the level of initiation. Specifically, BC1 RNA inhibited formation of the 48S preinitiation complex, i.e., recruitment of the small ribosomal subunit to the messenger RNA (mRNA). However, 48S complex formation that is independent of the eukaryotic initiation factor 4 (eIF4) family of initiation factors was found to be refractory to inhibition by BC1 RNA, a result that implicates at least one of these factors in the BC1 repression pathway. Biochemical experiments indicated a specific interaction of BC1 RNA with eIF4A, an RNA unwinding factor, and with poly(A)-binding protein. Both proteins were found enriched in synaptodendritic microdomains. Significantly, BC1-mediated repression was shown to be effective not only in cap-dependent translation initiation but also in eIF4-dependent internal initiation. The results suggest a functional role of BC1 RNA as a mediator of translational control in local protein synthesis in nerve cells.

Animals↗
PubMed2000

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.

Animals↗
PubMed1993

RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1.

RNA fingerprinting using arbitrarily primed PCR (RAP) samples an RNA population and allows the detection of differentially expressed genes between two or more populations. This method was applied to mink lung epithelial cells, which respond to treatment with transforming growth factor beta (TGF-beta) by undergoing cell cycle arrest at or near the G1/S-phase boundary. The steady-state abundances of approximately 200 RNAs were surveyed, a few of which displayed differential regulation in response to TGF-beta 1. Three products were isolated, cloned, and sequenced. One differentially regulated RNA corresponded to cyclin A, a gene known to be required for the progression of mammalian fibroblasts through S phase. Northern blot analysis confirmed that the cyclin A mRNA steady-state abundance decreased dramatically in response to a 24-hr TGF-beta 1 treatment and also in response to cell cycle arrest caused by contact inhibition. A second RAP product corresponded to a previously unknown 7.5-kb mRNA, the level of which decreased dramatically in response to TGF-beta 1 treatment. Unlike the cyclin A mRNA, the abundance of this transcript did not decrease in response to growth arrest induced by contact inhibition. A third RAP product corresponded to the mRNA for osteonectin, an extracellular matrix protein. The abundance of this mRNA increased at least 2-fold during TGF-beta 1 treatment. This observation is consistent with other reports of increases in extracellular matrix proteins during TGF-beta treatment. RAP should be able to identify many of the genes that change in steady-state expression during the cell cycle.

Animals↗
PubMed2006

Specific down-regulation of XIAP with RNA interference enhances the sensitivity of canine tumor cell-lines to TRAIL and doxorubicin.

BACKGROUND: Apoptosis resistance occurs in various tumors. The anti-apoptotic XIAP protein is responsible for inhibiting apoptosis by reducing caspase-3 activation. Our aim is to evaluate whether RNA inhibition against XIAP increases the sensitivity of canine cell-lines for chemotherapeutics such as TRAIL and doxorubicin. We used small interfering RNA's (siRNA) directed against XIAP in three cell-lines derived from bile-duct epithelia (BDE), mammary carcinoma (P114), and osteosarcoma (D17). These cell-lines represent frequently occurring canine cancers and are highly comparable to their human counterparts. XIAP down-regulation was measured by means of quantitative PCR (Q-PCR) and Western blotting. The XIAP depleted cells were treated with a serial dilution of TRAIL or doxorubicin and compared to mock- and nonsense-treated controls. Viability was measured with a MTT assay. RESULTS: All XIAP siRNA treated cell-lines showed a mRNA down-regulation over 80 percent. Western blot analysis confirmed mRNA measurements. No compensatory effect of IAP family members was seen in XIAP depleted cells. The sensitivity of XIAP depleted cells for TRAIL was highest in BDE cells with an increase in the ED50 of 14-fold, compared to mock- and nonsense-treated controls. The sensitivity of P114 and D17 cell-lines increased six- and five-fold, respectively. Doxorubicin treatment in XIAP depleted cells increased sensitivity in BDE cells more than eight-fold, whereas P114 and D17 cell-lines showed an increase in sensitivity of three- and five-fold, respectively. CONCLUSION: XIAP directed siRNA's have a strong sensitizing effect on TRAIL-reduced cell-viability and a smaller but significant effect with the DNA damaging drug doxorubicin. The increase in efficacy of chemotherapeutics with XIAP depletion provides the rationale for the use of XIAP siRNA's in insensitive canine tumors.

Animals↗
PubMed2006

Cultured lymphocytes from autistic children and non-autistic siblings up-regulate heat shock protein RNA in response to thimerosal challenge.

There are reports suggesting that some autistic children are unable to mount an adequate response following exposure to environmental toxins. This potential deficit, coupled with the similarity in clinical presentations of autism and some heavy metal toxicities, has led to the suggestion that heavy metal poisoning might play a role in the etiology of autism in uniquely susceptible individuals. Thimerosal, an anti-microbial preservative previously added routinely to childhood multi-dose vaccines, is composed of 49.6% ethyl mercury. Based on the levels of this toxin that children receive through routine immunization schedules in the first years of life, it has been postulated that thimerosal may be a potential triggering mechanism contributing to autism in susceptible individuals. One potential risk factor in these individuals may be an inability to adequately up-regulate metallothionein (MT) biosynthesis in response to presentation of a heavy metal challenge. To investigate this hypothesis, cultured lymphocytes (obtained from the Autism Genetic Resource Exchange, AGRE) from autistic children and non-autistic siblings were challenged with either 10 microM ethyl mercury, 150 microM zinc, or fresh media (control). Following the challenge, total RNA was extracted and used to query "whole genome" DNA microarrays. Cultured lymphocytes challenged with zinc responded with an impressive up-regulation of MT transcripts (at least nine different MTs were over-expressed) while cells challenged with thimerosal responded by up-regulating numerous heat shock protein transcripts, but not MTs. Although there were no apparent differences between autistic and non-autistic sibling responses in this very small sampling group, the differences in expression profiles between those cells treated with zinc versus thimerosal were dramatic. Determining cellular response, at the level of gene expression, has important implications for the understanding and treatment of conditions that result from exposure to neurotoxic compounds.

Anti-Infective Agents, Local↗
PubMed1990

Activating-transcription-factor (ATF) regulates human 7S L RNA transcription by RNA polymerase III in vivo and in vitro.

The gene-external part of the human 7S L promoter was analyzed by transcription in vitro and in vivo. Compared to the wild type promoter (-178), a -66 5'deletion mutant revealed full activity in vitro but was inefficiently transcribed in vivo. Further deletion to -37 reduced template activity to 50% in vitro and to basal level expression in vivo (below 5%). A DNase I footprint observed around position -50 protected an ATF-like binding site ('TGACGT'). With respect to 7S L transcription regulation, the functionality of this ATF-like binding site was confirmed in competition experiments and by mutation analysis. Furthermore, S100 extracts of cells pretreated with forskolin in vivo to induce the cAMP system, revealed significantly increased transcription of 7S L RNA in vitro, with no effect on a 7S K RNA gene, lacking such an ATF binding site. Thus, the 7S L RNA gene too is controlled by a regulatory element originally defined in class II promoters and represents another rare example where a specific type of transcription regulation in vivo can be mimicked with cell-free extracts in vitro.

Activating Transcription Factors↗
PubMed1994

Regulation of adenovirus alternative RNA splicing at the level of commitment complex formation.

The adenovirus late region 1 (L1) represents an example of an alternatively spliced gene where one 5' splice site is spliced to two alternative 3' splice sites, to produce two mRNAs; the 52,55K and IIIa mRNAs, respectively. Accumulation of the L1 mRNAs is temporally regulated during the infectious cycle. Thus, the proximal 3' splice site (52,55K mRNA) is used at all times during the infectious cycle whereas the distal 3' splice site (IIIa mRNA) is used exclusively late in infection. Here we show that in vitro splicing extracts prepared from late adenovirus-infected cells reproduces the virus-induced temporal shift from proximal to distal 3' splice site selection in L1 pre-mRNA splicing. Two stable intermediates in spliceosome assembly have been identified; the commitment complex and the pre-spliceosome (or A complex). We show that the transition in splice site activity in L1 alternative splicing results from an increase in the efficiency of commitment complex formation using the distal 3' splice site in extracts prepared from late virus-infected cells combined with a reduction of the efficiency of proximal 3' splice site splicing. The increase in commitment activity on the distal 3' splice site is paralleled by a virus-induced increase in A complex formation on the distal 3' splice site. Importantly, the virus-induced shift from proximal to distal L1 3' splice site usage does not require cis competition between the 52,55K and the IIIa 3' splice sites, but rather results from the intrinsic property of the two 3' splice sites which make them respond differently to factors in extracts prepared from virus-infected cells.

Adenoviruses, Human↗