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At least 73 records · Page 4Linked to original sources
PubMed2006

RNAi-based screening of the human kinome identifies Akt-cooperating kinases: a new approach to designing efficacious multitargeted kinase inhibitors.

Tumors comprise genetically heterogeneous cell populations, whose growth and survival depend on multiple signaling pathways. This has spurred the development of multitargeted therapies, including small molecules that can inhibit multiple kinases. A major challenge in designing such molecules is to determine which kinases to inhibit in each cancer to maximize efficacy and therapeutic index. We describe an approach to this problem implementing RNA interference technology. In order to identify Akt-cooperating kinases, we screened a library of kinase-directed small interfering RNAs (siRNAs) for enhanced cancer cell killing in the presence of Akt inhibitor A-443654. siRNAs targeting casein kinase I gamma 3 (CSNK1G3) or the inositol polyphosphate multikinase (IPMK) significantly enhanced A-443654-mediated cell killing, and caused decreases in Akt Ser-473 and ribosomal protein S6 phosphorylation. Small molecules targeting CSNK1G3 and/or IPMK in addition to Akt may thus exhibit increased efficacy and have the potential for improved therapeutic index.

Antineoplastic Agents↗
PubMed1998

Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis.

In Xenopus oocytes, progesterone stimulates the cytoplasmic polyadenylation and resulting translational activation of c-mos mRNA, which is necessary for the induction of oocyte maturation. Although details of the biochemistry of polyadenylation are beginning to emerge, the mechanism by which 3' poly(A) addition stimulates translation initiation is enigmatic. A previous report showed that polyadenylation induced cap-specific 2'-O-methylation, and suggested that this 5' end modification was important for translational activation. Here, we demonstrate that injected c-mos RNA undergoes polyadenylation and cap ribose methylation. Inhibition of this methylation by S-isobutylthioadenosine (SIBA), a methyltransferase inhibitor, has little effect on progesterone-induced c-mos mRNA polyadenylation or general protein synthesis, but prevents the synthesis of Mos protein as well as oocyte maturation. Maturation can be rescued, however, by the injection of factors that act downstream of Mos, such as cyclin A and B mRNAs. Most importantly, we show that the translational efficiency of injected mRNAs containing cap-specific 2'-O-methylation (cap I) is significantly enhanced compared to RNAs that do not contain the methylated ribose (cap 0). These results suggest that cap ribose methylation of c-mos mRNA is important for translational recruitment and for the progression of oocytes through meiosis.

Animals↗
PubMed2006

Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses.

OBJECTIVE: Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages. METHODS AND RESULTS: Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor alpha. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1beta and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1alpha and -1beta and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized-low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation. CONCLUSIONS: NR4A-factors are expressed in human atherosclerotic lesion macrophages and reduce human macrophage lipid loading and inflammatory responses, providing further evidence for a protective role of NR4A-factors in atherogenesis.

Atherosclerosis↗
PubMed2004

Activation of the hepatocyte growth factor (HGF)-Met system in papillary thyroid cancer: biological effects of HGF in thyroid cancer cells depend on Met expression levels.

Met, the receptor for hepatocyte growth factor (HGF), is overexpressed in approximately 90% papillary thyroid carcinomas. To investigate the role of the HGF-Met system in these tumors, we examined HGF and Met expression in a variety of primary cultures, normal or malignant thyroid cells, and tissue specimens and analyzed the different HGF effects (promotion of mitogenesis, branching morphogenesis, and cell motility and invasion). In cancer specimens, HGF was produced at high levels by tumor stromal cells, and Met was constitutively phosphorylated in malignant cells. No HGF production was found in a panel of malignant thyroid cancer cells. Biological effects of HGF were examined in papillary cancer cell cultures with either high or low Met expression. High-Met cells were more sensitive to the growth effect of HGF (ED50 = 3-5 ng/ml and 10-15 ng/ml in high- or low-Met cells, respectively). Moreover, only high-Met cells underwent branching morphogenesis in response to HGF. In contrast, high-Met cells showed a reduced migration. Met down-regulation by small interfering RNAs resulted in enhanced cell migration and abrogation of branching morphogenesis in response to HGF. Conversely, Met overexpression impaired cell migration while favoring branching morphogenesis and cell adherence to substrate. These data suggest that both Met and HGF are overexpressed in papillary thyroid carcinomas, that Met is frequently activated in these carcinomas and may favor tumor growth, and that the abundance of Met expression may differently regulate cell growth, morphogenesis, and migration in response to HGF.

Carcinoma, Papillary↗
PubMed2000

Cap-independent translational enhancement of turnip crinkle virus genomic and subgenomic RNAs.

The presence of translational control elements and cap structures has not been carefully investigated for members of the Carmovirus genus, a group of small icosahedral plant viruses with positive-sense RNA genomes. In this study, we examined both the 5' and 3' untranslated regions (UTRs) of the turnip crinkle carmovirus (TCV) genomic RNA (4 kb) as well as the 5' UTR of the coat protein subgenomic RNA (1.45 kb) for their roles in translational regulation. All three UTRs enhanced translation of the firefly luciferase reporter gene to different extents. Optimal translational efficiency was achieved when mRNAs contained both 5' and 3' UTRs. The synergistic effect due to the 5'-3' cooperation was at least fourfold greater than the sum of the contributions of the individual UTRs. The observed translational enhancement of TCV mRNAs occurred in a cap-independent manner, a result consistent with the demonstration, using a cap-specific antibody, that the 5' end of the TCV genomic RNA was uncapped. Finally, the translational enhancement activity within the 5' UTR of 1.45-kb subgenomic RNA was shown to be important for the translation of coat protein in protoplasts and for virulent infection in Arabidopsis plants.

3' Untranslated Regions↗
PubMed1993

Cleavage of full-length beta APP mRNA by hammerhead ribozymes.

The sequences surrounding the first 5'GUC3' in the mRNA encoding the Alzheimer amyloid peptide precursor (beta APP) were used to construct a pair of transacting hammerhead ribozymes. Each ribozyme contained the conserved core bases of the hammerhead motif found in the positive strand of satellite RNA of tobacco ringspot virus [(+)sTRSV] and two stems, 7 and 8 bases long, complementary to the target, beta APP mRNA. However, one of the ribozyme cleaving strands was lengthened at its 3' end to include the early splicing and polyadenylation signal sequences of SV40 viral RNA. This RNA, therefore, more closely mimics transcripts produced by RNA polymerase II from eucaryotic expression vectors in vivo. RNA, prepared by run-off transcription of cDNA oligonucleotide or plasmid constructs containing a T7 RNA polymerase promoter was used to characterize several properties of the cleavage reaction. In the presence of both ribozyme cleaving strands magnesium-ion dependent cleavage of a model 26 base beta APP substrate RNA or full-length beta APP-751 mRNA was observed at the hammerhead consensus cleavage site. Neither ribozyme was active against non-message homologs of beta APP mRNA, nor was cleavage detected when point mutations were made in the conserved core sequences. However, the kcat/Km at 37 degrees C in 10 mM Mg+2 of the longer ribozyme was reduced twenty-fold when model and full-length substrates were compared. The use of short deoxyoligonucleotides (13-17 mers) that bind upstream of the ribozyme was found to enhance the rate of cleavage of the full-length but not beta APP model substrate RNAs. The rate of enhancement depended on both the length of the deoxyoligonucleotide used as well as its site of binding with respect to the ribozyme. These data demonstrate the utility of ribozymes to cleave target RNAs in a catalytic, site-specific fashion in vitro. Direct comparison of the efficiency of different ribozyme constructs and different modulating activities provide an experimental strategy for designing more effective ribozymes for therapeutic purposes.

Amyloid beta-Protein Precursor↗
PubMed1994

Enhanced competitiveness of tomato bushy stunt virus defective interfering RNAs by segment duplication or nucleotide insertion.

We have analyzed atypical tomato bushy stunt virus defective interfering (DI) RNA species which accumulated during a passage series in protoplasts. We present a rationale for the order of appearance of these molecules and show, using competition assays, that either segment duplication or single nucleotide insertion can enhance DI RNA competitiveness. Possible mechanisms for the introduction of the modifications observed in these DI RNAs are discussed.

Base Sequence↗
PubMed2000

Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation.

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

Base Sequence↗
PubMed2004

Overexpression of Dicer enhances RNAi-mediated gene silencing by short-hairpin RNAs (shRNAs) in human cells.

RNA interference (RNAi) is a process in which small-interfering RNA (siRNA) induces a sequence-dependent degradation of mRNA. In recent years, vector-based siRNA expression systems have been developed by several groups. In these systems, short-hairpin RNAs (shRNAs) have been widely chosen for first transcriptional products which are processed to siRNAs by Dicer-dependent cleavage. In this study, we demonstrate that overexpression of Dicer enhances the effect of shRNA-dependent gene silencing in mammalian cells. Therefore, these results indicate that Dicer-dependent cleavage of shRNAs is an important step for shRNA-mediated gene silencing.

Animals↗
PubMed1994

Gene regulation of cyclic AMP-dependent protein kinase subunits (C alpha, beta; RI alpha, beta and RII alpha, beta) in rat facial motoneurons after nerve transection.

In this study we investigated the changes in gene expressions of catalytic (C alpha, beta) and regulatory (RI alpha, beta and RII alpha, beta) subunits of cAMP-dependent protein kinase (PKA) in axotomized facial motoneurons of the rat. Nerve transection induced changes in the expression of C subunit messenger RNAs and RII subunit messenger RNAs. Control facial motoneurons had a high expression of both C alpha and C beta subunit messenger RNAs, but their expression declined after axotomy. The decrease was most pronounced at postoperative week 2 and returned to basal level within postoperative week 4. In contrast, the expression of both RII alpha and beta subunit messenger RNAs, which were low in control facial motoneurons, was increased after axotomy. Enhancement of RII subunits messenger RNAs was apparent during postoperative weeks 1 and 3, and then returned to the basal level. RI alpha, beta subunits messenger RNAs were strongly expressed in normal facial motoneurons, but were not clearly influenced by axotomy. These results indicate an attenuation of total PKA activity in axotomized facial motoneurons. Furthermore, such gene regulation may imply a change of the targets for PKA in facial motoneurons during the process of neurite regeneration.

Animals↗
PubMed1987

Regulated expression of human atrial natriuretic polypeptide gene in mouse L cells.

To investigate whether the human atrial natriuretic polypeptide (hANP) gene is responsive to glucocorticoid, we co-introduced the hANP gene (with SV40 enhancer) with HSV-tk gene into mouse tk- L cells. The transformants with hANP gene with SV40 enhancer expressed hANP specific RNAs. The administration of 1 microM dexamethasone reduced the expressed hANP specific RNAs, especially those that had a physiological initiation site. These results suggest that the hANP gene is really a glucocorticoid responsive gene and may be negatively regulated by glucocorticoid.

Animals↗
PubMed2002

The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells.

The solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5" boundary area of the erythroid beta-globin locus control region (beta-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. Functional studies utilizing the green fluorescent protein (GFP) gene as the reporter in transfection experiments show that the U3 region of the LTRs possesses strong enhancer activity in embryonic cells of widely different tissue origins and in adult cells of blood lineages. In both the genomic LTRs of embryonic placental cells and erythroid K562 cells and transfected LTRs of recombinant GFP plasmids in K562 cells, the U3 enhancer activates synthesis of RNAs that are initiated from a specific site 25 bases downstream of the AATAAA (TATA) motif in the U3 promoter. A second AATAAA motif in the R region does not serve as the TATA box or as the polyadenylation signal. The LTR-initiated RNAs extend through the R and U5 regions into the downstream genomic DNA. The results suggest that the ERV-9 LTR-initiated transcription process may modulate transcription of the associated gene loci in embryonic and hematopoietic cells.

Animals↗
PubMed2006

Induction of interferon synthesis by the PKR-inhibitory VA RNAs of adenoviruses.

In virus-infected cells, double-stranded RNA (dsRNA) activates the transcription factor interferon (IFN) regulatory factor-3 (IRF-3), which stimulates type I IFN (IFN-alpha/beta) gene expression. In addition, dsRNA activates the enzyme RNA-activated protein kinase (PKR), which phosphorylates the eukaryotic initiation factor 2alpha (eIF2beta), thereby inhibiting mRNA translation. Adenoviruses express highly structured RNA molecules termed VA RNAs (VA(I)/VA(II)) known to specifically inhibit PKR. As PKR impairs expression from transfected cDNA constructs, plasmids encoding VA RNAs are widely used as enhancers of transgene expression. Here, we describe induction of IFN synthesis as a novel feature of VA RNAs. Transfection of a VA(I)/VA(II)-expressing plasmid was found to induce type I IFN production, resulting in activation of IFN-dependent genes, such as IFN-stimulated gene 56 (ISG56) or MxA, and the establishment of an antiviral state in transfected cells. Curiously, VA RNAs did not activate IRF-3, suggesting an alternative pathway of IFN induction. These data may be considered when using genetically modified adenoviruses as therapeutic agents and suggest caution in choosing VA RNA constructs as a means to increase expression of a gene of interest.

Adenoviridae↗
PubMed2004

Regulation of relative abundance of arterivirus subgenomic mRNAs.

The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5'- and 3'-coterminal nested set with the viral genome. Their 5' common leader sequence is derived from the genomic 5'-proximal region. Fusion of sg RNA leader and "body" segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.

Equartevirus↗
PubMed2003

Enhanced gene silencing by the application of multiple specific small interfering RNAs.

Small interfering RNA duplexes (siRNA) induce gene silencing in various eukaryotic cells, although usually in an incomplete manner. Using chemically synthesized siRNAs targeting the HIV-1 co-receptor CXCR4 or the apoptosis-inducing Fas-ligand (FasL), co-transfection of cells with two or more siRNA duplexes targeting different sites on the same mRNA resulted in an enhanced gene silencing compared with each single siRNA. This was shown in the down-regulation of protein and mRNA expression, and functionally in the inhibition of CXCR4-mediated HIV infection and of FasL-mediated cell apoptosis. Transfection efficiency determined for the FasL-specific siRNAs was dose-dependent and varied among the siRNAs tested, but was not the main reason for the enhanced gene silencing.

Apoptosis↗
PubMed1996

Development of an in vitro mRNA degradation assay utilizing extracts from HIV-1- and SIV-infected cells.

We previously demonstrated that cellular mRNAs are degraded in CD4 positive lymphocytes infected by the human immunodeficiency virus, HIV-1, but not in cells infected by the simian lentivirus, SIV. To begin to define the molecular mechanisms underlying this RNA degradation, we have established an in vitro RNA degradation assay utilizing extracts from both infected and uninfected cells. We found that in vitro transcribed, 32P-radiolabeled actin RNA was degraded in extracts prepared from CEM, CEMx174, and C8166 cells which were infected with HIV-1. Minimal actin RNA degradation was observed in extracts prepared from uninfected cells. Similarly little degradation was observed in cell-free extracts prepared from SIV-infected cells. To determine if viral RNA sequences could impart enhanced stability to cellular RNAs in our in vitro assay, we prepared radiolabeled RNAs that contained selected viral RNA determinants. One such RNA contained the HIV-1 specific TAR (transactivating region) sequence (nucleotides 1-111) appended to a reporter CAT RNA. Like the cellular actin RNA, these TARCAT RNAs were degraded in HIV-1-infected cell extracts, but not in extracts from uninfected cells or extracts prepared from SIV-infected cells. In contrast, an RNA containing only authentic HIV-1 sequences comprising TAR and gag sequences was more stable than actin RNA in HIV-1-infected extracts. These results, taken together, suggest that the in vitro assay reproduces events that occur in vivo and provide a starting point for identifying the factor responsible for cellular RNA degradation in HIV-1-infected cells.

Actins↗
PubMed1997

Post-transcriptional gene silencing in plants.

Overexpression of chimeric transgenes in plants can trigger post-transcriptional gene silencing that is dependent on epigenetic information and physiological conditions. The current view is that unproductive RNA serves as a crucial signal for gene silencing, although direct evidence is lacking for this theory. A signalling cascade then leads to strongly enhanced turnover of all RNAs that share a critical degree of sequence similarity. The molecular details of the mechanism are, however, insufficiently understood to explain the phenomenon completely and to comprehend its biological significance.

5-Methylcytosine↗
PubMed1996

Cell-cycle related regulation of poly(A) polymerase by phosphorylation.

The poly(A) tail found on almost all eukaryotic messenger RNAs is important in enhancing translation initiation and determining mRNA stability. Control of poly(A)-tail synthesis thus has the potential to be a key regulatory step in gene expression and is indeed known to be important during early development in many organisms. To study a possible basis for such regulation, we examined phosphorylation of poly(A) polymerase (PAP) by p34(cdc2)/cyclin B (maturation/mitosis-promoting factor, MPF). We show here that PAP can be phosphorylated in vivo and in vitro by MPF. Consistent with this, PAP becomes hyperphosphorylated both during meiotic maturation of Xenopus laevis oocytes and in HeLa cells arrested at M phase, times in the cell-cycle when MPF is known to be active. We show further that hyperphosphorylation by MPF dramatically reduces the activity of purified PAP, and that PAP isolated from mitotic HeLa cells is similarly inhibited by hyperphosphorylation. This repression probably contributes to the well established reductions in poly(A)+ RNA and/or protein synthesis known to occur in M-phase cells.

Animals↗