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At least 55 records · Page 3Linked to original sources
PubMed1989

Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells.

We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.

Animals↗
PubMed1996

Enhanced cytochemical detection of viral proteins and RNAs using double-sided labeling and light microscopy.

We have developed a double-sided labeling technique for detecting viral proteins or RNAs in plastic-embedded leaf tissue by immunocytochemistry or in situ hybridization, respectively, and light microscopy. The signal from the target was enhanced by double-sided labeling when compared with single-sided labeling because sections were submerged in labeling solutions with both sides accessible to antibodies or complementary RNAs. The additional label was visible during microscopic analysis. Background signal was decreased since the tissue was probed and washed under conditions where folds and creases in the tissue were minimized. This technique uses the same equipment and chemicals as for single-sided labeling, and thus adjustments for reagent expenditures are not necessary. The procedure should be applicable to animal and plant tissue.

Animals↗
PubMed1992

Evolution of intestinal apolipoprotein B mRNA editing. Chicken apolipoprotein B mRNA is not edited, but chicken enterocytes contain in vitro editing enhancement factor(s).

Mammalian intestinal apolipoprotein B (apoB) messenger RNA (mRNA) undergoes posttranscriptional editing, changing codon 2153 from CAA in apoB100 mRNA to an in-frame translational stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB cDNA contains a CAA codon at the corresponding site and apoB mRNA from chicken enterocytes, kidney, and liver is unedited. The cDNA sequence of chicken apoB spanning the edited base is divergent from mammalian apoB cDNA sequence, with 70% homology over the conserved 29-nucleotide sequence (6662-6690) flanking codon 2153. Efficient in vitro editing of both human and rat, but not chicken, synthetic apoB RNA was achieved using rat enterocyte S-100 extracts. By contrast, chicken enterocyte S-100 extracts failed to edit chicken, rat, or human synthetic apoB RNA. Mixing experiments, however, revealed that chicken enterocyte S-100 extracts enhance the in vitro editing activity of rat, pig, and human enterocyte S-100 extracts upon homologous RNAs. The editing enhancement activity of chicken enterocyte S-100 extracts is tissue-specific, heat-sensitive, substrate-saturable, and sensitive to proteinase K, but resistant to micrococcal nuclease. The activity was partially purified by Q-Sepharose chromatography and has an average molecular mass of 49 kDa when analyzed by gel filtration chromatography. We conclude that the evolutionary adaptation of intestinal apoB mRNA editing requires both a requisite RNA motif and tissue-specific factors which mediate the site-specific modification.

Animals↗
PubMed1993

Enhanced production of morbillivirus gene-specific RNAs following induction of the cellular stress response in stable persistent infection.

Previous in vitro work demonstrated the incorporation of the major inducible 70k heat shock protein (i.e., 72k HSP) into the biologically active light nucleocapsid (L-NC) variant of canine distemper virus (CDV). Here, in vitro induction of the cellular stress response, characterized by elevated cytoplasmic and intranuclear 72k HSP, enhanced L-NC expression in mink lung cells supporting stable persistent infection by raccoon-origin CDV. Increases in L-NC were correlated to increased viral RNA production in cell-free transcriptional assays. The enhanced production of viral transcripts within infected cells following stress response induction was confirmed by slot blot and Northern blot analysis of total cellular RNA and was reflected in increased total viral protein production. Post-shock increases in viral fusion (F) gene transcripts and F protein were associated with dramatic increases in viral cytopathic effect. Modest induction of cell-free infectious viral progeny was also documented. A similar effect of the cellular stress response upon viral protein expression, cytopathic effect, and cell-free infectious progeny release was demonstrated in murine neuroblastoma cells persistently infected with a canine CDV isolate. Alterations of the persistent viral phenotype were independent of the specific mechanism of stress-response induction (i.e., heat or sodium arsenite), supporting the role of the stress response and not a particular stressor in mediating these changes. These results document the ability of the cellular environment to alter persistent viral RNA metabolism, thereby altering the infection phenotype.

Animals↗
PubMed2003

An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs.

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.

Base Sequence↗
PubMed2005

Coat protein enhances translational efficiency of Alfalfa mosaic virus RNAs and interacts with the eIF4G component of initiation factor eIF4F.

The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc-AMV), a poly(A) tail of 50 residues [Luc-poly(A)] or a short vector-derived sequence (Luc-control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc-poly(A) or Luc-control, but strongly stimulated Luc expression from Luc-AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc-AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc-AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.

3' Untranslated Regions↗
PubMed2004

Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs.

Plasmids or viral vectors that express short hairpin RNAs (shRNAs) have emerged as important tools for the stable inhibition of specific genes by RNA interference. shRNAs are structural and functional homologs of pre-microRNAs, intermediates in the production of endogenously encoded microRNAs (miRNAs). Therefore, overexpressed shRNAs could inhibit miRNA function by competing for a limiting level of one or more factors involved in miRNA biogenesis or function. Here, we demonstrate that overexpressed shRNAs can saturate the activity of endogenous Exportin 5, a factor required for nuclear export of both shRNAs and pre-miRNAs. While shRNA overexpression can therefore inhibit miRNA function, simultaneous overexpression of Exportin 5 reverses this effect. Moreover, Exportin 5 overexpression can significantly enhance RNA interference mediated by shRNAs. These data have implications for the future clinical utilization of shRNAs and also provide a simple method to enhance RNA interference by shRNAs in culture.

Active Transport, Cell Nucleus↗
PubMed1998

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2.

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.

Alternative Splicing↗
PubMed2005

Biomembrane-permeable and Ribonuclease-resistant siRNA with enhanced activity.

Small interfering RNAs (siRNA) could be the ideal inhibitor of specific gene expression if they could be delivered efficiently to their targets. Although siRNAs cannot by themselves cross cell membranes, they can enter cells with the help of transfection reagents via facilitated transport. Inside cells, however, they can still be hydrolyzed by cytoplasmic RNases before reaching their target. Here we show that poly-2'- O-(2,4-dinitrophenyl)-siRNA (DNP-siRNA) can by itself diffuse into mammalian cells and stay in the cells for several days, with no detectable degradation. Using the DNP-RNA targeting to the insulin-like growth factor receptor (IGF-IR) as an example, we demonstrate that the efficacy of this DNP-siRNA for inhibiting the growth of three different types of tumor cells is several-fold higher than that of the corresponding native siRNA. Similar results were obtained from Western blotting assay. As expected, the control siRNAs with mismatched, scrambled, and reverse sequences are all inactive. These results illustrate that both the biomembrane permeability and intracellular stability of siRNA can be greatly improved by DNP derivatization without loss of its sequence specificity.

2,4-Dinitrophenol↗
PubMed2026

Enhanced CRISPR-Cas3-mediated genome editing using circularized crRNAs.

Type I-E CRISPR-Cas3 represents a genome-editing technology in which large deletions averaging several kilobases are introduced in target regions. However, its genome-editing efficiency varies considerably across targets and cell types, making it difficult to achieve consistent results. Here, we investigated the efficacy and stability of circularized CRISPR RNAs (ccrRNAs) to enhance CRISPR-Cas3-mediated genome editing in human cells. Using in vitro single-strand DNA cleavage assays, we demonstrated that ccrRNA induces Cascade complex formation. Significant genome-editing activity targeting the EMX1 and B2M genes was observed in cellular assays using K562 cells. Long-read sequencing identified large-scale deletion mutations at the target loci and no detectable off-target effects using ccrRNA. Furthermore, ccrRNAs exhibited extended intracellular stability compared with that for linear crRNAs, resulting in an enhanced editing efficiency. These results demonstrate that ccrRNAs enable stable, efficient, and highly specific genome editing and support the broader application of the long-range deletion system.

CRISPR-Cas3↗
PubMedDate not supplied

Enhancement of in-vitro translation of eukaryotic RNAs by cyclic AMP.

Addition of cyclic AMP to normal rabbit reticulocyte lysate brings about substantial increase in protein synthesis programmed by both nuclear and polysomal eukaryotic RNAs. The increase in synthesis is largely nonspecific and the in-vitro translation system is rendered viable for longer periods of time than in the absence of cyclic AMP. This stimulation of translation could be due to the inhibition of protein kinases that are initially activated by secondary structure of RNAs. However, the ability of cyclic AMP to further stimulate methyl mercury or heat denatured RNA indicates that additional mechanisms may be involved in the enhancement of translation.

Animals↗
PubMed1984

Effect of interferon on integrity of vaccinia virus and ribosomal RNA in infected cells.

The state of vaccinia and ribosomal RNAs in IFN-treated, vaccinia virus-infected mouse L cells grown in suspension is examined. In these cells a drastic inhibition (approximately 90%) of both viral and cellular protein synthesis occurs after virus infection of IFN-treated cells. The findings show that (1) primary transcription of vaccinia virus is not impaired by IFN, but is rather enhanced; (2) viral RNAs produced in IFN-treated, infected cells are predominantly of the early class; (3) these viral RNAs can be translated in a cell-free protein-synthesizing system; (4) in IFN-treated, infected cells there is extensive cleavage of 28 and 18 S rRNA at early times post infection, resulting in a characteristic cleavage pattern; (5) cleavage of rRNA is viral RNA dependent. The results indicate that in this virus-cell system the IFN-mediated inhibition of vaccinia and cellular protein synthesis is correlated with an alteration in ribosomal integrity.

Animals↗
PubMed2005

Composition and sequence-dependent binding of RNA to the nucleocapsid protein of Moloney murine leukemia virus.

All retroviruses package two copies of their genomes during virus assembly, both of which are required for strand transfer-mediated recombination during reverse transcription. Genome packaging is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and an RNA packaging signal, located near the 5' end of the genome, called Psi. We recently discovered that the NC protein of the Moloney murine leukemia virus (MLV) can bind with high affinity to conserved UCUG elements within the MLV packaging signal [D'Souza, V., and Summers, M. F. (2004) Nature 431, 586-590]. Selective binding to dimeric RNA is regulated by a conformational RNA switch, in which the UCUG elements are sequestered by base pairing in the monomeric RNA and do not bind NC, but become exposed for NC binding upon dimerization. Dimerization-dependent structural changes occur in other regions of the Psi-site, exposing guanosine-containing segments that might also bind NC. Here we demonstrate that short RNAs containing three such sequences, ACAG, UUUG, and UCCG, can bind NC with significant affinity (K(d) = 94-315 nM). Titration experiments with oligoribonucleotides of varying lengths and compositions, combined with NMR-based structural studies, reveal that binding is strictly dependent on the presence of an unpaired guanosine, and that relative binding affinities can vary by more than 1 order of magnitude depending on the nature of the three upstream nucleotides. Binding is enhanced in short RNAs containing terminal phosphates, indicating that electrostatic interactions contribute significantly to binding. Our findings extend a previously published model for genome recognition, in which the NC domains of assembling Gag molecules interact with multiple X(i-3)-X(i-2)-X(i-1)-G(i) elements (X is a variable nucleotide) that appear to be preferentially exposed in the dimeric RNA.

Base Composition↗
PubMed2000

Targeting a Pneumocystis carinii group I intron with methylphosphonate oligonucleotides: backbone charge is not required for binding or reactivity.

Pneumocystis carinii is a mammalian pathogen that contains a self-splicing group I intron in its large subunit rRNA precursor. We report the binding of methylphosphonate/DNA chimeras and neutral methylphosphonate oligonucleotides to a ribozyme that is a truncated form of the intron. At 15 mM Mg(2+), the nuclease-resistant all-methylphosphonate hexamer, d(AmTmGmAmCm)rU, with a sequence that mimics the 3' end of the precursor's 5' exon, binds with a dissociation constant of 272 nM. The hexamer's dissociation constant for binding by base-pairing alone to the ribozyme's binding site sequence is 8.3 mM. Thus there is a 30 000-fold binding enhancement by tertiary interactions (BETI), which is close to the 60 000-fold enhancement previously observed with the all-ribo hexamer, r(AUGACU). Evidently, backbone charge and 2' hydroxyl groups are not required for BETI. At 3-15 mM Mg(2+), the all-methylphosphonate and DNA oligonucleotides trans-splice to a truncated form of the rRNA precursor, but do not compete with cis-splicing when pG is present. These results suggest that uncharged or partially charged backbones may be used to design therapeutics to target RNAs through binding enhancement by tertiary interactions and suicide inhibition strategies.

Binding Sites↗
PubMed1999

The antiviral enzymes PKR and RNase L suppress gene expression from viral and non-viral based vectors.

Expression of transfected genes is shown to be suppressed by two intracellular enzymes, RNase L and protein kinase PKR, which function in interferon-treated cells to restrict viral replication. RNase L(-/-) or PKR(-/-) murine embryonic fibroblasts produced enhanced levels of protein from transfected genes compared with wild-type cells. Increased expression of exogenous genes in RNase L(-/-) cells correlated with elevated levels of mRNA and thus appeared to be due to enhanced mRNA stability. Plasmid encoding adenovirus VA RNAs was able to further enhance accumulation of the exogenous gene transcript and protein, even in cells lacking PKR. In contrast to the increased expression of transfected genes in cells lacking RNase L or PKR, expression of endogenous host genes was unaffected by the absence of these enzymes. In addition, a dominant-negative PKR mutant improved expression from a conventional plasmid vector and from a Semliki Forest virus derived, self-replicating vector. These results indicate that viral infections and transfections produce similar stress responses in mammalian cells and suggest strategies for selectively increasing expression of exogenous genes.

Adenoviridae↗
PubMed1994

Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements.

In the cytoplasm of oocytes and early embryos, addition of poly(A) to mRNAs can activate their translation. We demonstrate that despite many differences between poly(A) addition in the cytoplasm and nucleus, these two forms of polyadenylation may involve identical trans-acting factors. Nuclear polyadenylation requires the sequence AAUAAA, the AAUAAA-binding cleavage and polyadenylation specificity factor (CPSF), and a poly(A) polymerase (PAP). We show that CPSF and PAP, purified from calf thymus, exhibit the same sequence specificity observed in the cytoplasm during frog oocyte maturation, requiring both AAUAAA and a proximal U-rich sequence. The enhanced polyadenylation of RNAs containing U-rich sequences is caused by their increased affinity for CPSF. Frog nuclear polyadenylation factors display cytoplasmic sequence specificity when dilute, suggesting that a difference in their concentrations in the nucleus and cytoplasm underlies the different sequence specificities in the two compartments. Because polyadenylation in extracts prepared from oocytes before maturation is stimulated by addition of CPSF, the onset of polyadenylation during early development may be attributable to the activation or synthesis of a CPSF-like factor. We suggest that sequences upstream of AAUAAA that are required for cleavage and polyadenylation of certain pre-mRNAs in the nucleus may be functionally equivalent to the upstream, U-rich sequences that function in the cytoplasm, enhancing CPSF binding. We propose that CPSF and PAP comprise a core polyadenylation apparatus in the cytoplasm of oocytes and early embryos.

Animals↗