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At least 289 records · Page 16Linked to original sources
PubMed1988

Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA.

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.

Acetyltransferases↗
PubMed2005

Nested genes: biological implications and use of AFM for analysis.

A "nested" gene is located within the boundaries of a larger gene, often within an intron and in the opposite orientation. Such structures are common in bacteria and viruses, but have also been described in higher species as diverse as Drosophila and humans. Expression of nested and host genes may be simultaneously up-regulated due to use of common enhancers, or down-regulated through steric hindrance or interference caused by annealing of the complementary RNAs, leading to degradation. Methods for RNA analysis such as RT-PCR and in situ hybridization reveal the presence of specific mRNAs, but do not address regulation of expression within a single cell at a single genetic locus. Atomic force microscopy is a relatively new technology, which allows visualization of the movement of an RNA polymerase along a DNA template. The potential of this technology includes a greater molecular understanding of cellular decision making processes, leading to enhanced opportunities to intervene in disease progression through use of novel treatment modalities.

Animals↗
PubMed1994

FK506 enhances fibrogenesis in in vitro and in vivo models of liver fibrosis in rats.

BACKGROUND/AIMS: The immunosuppressant FK506 is undergoing clinical trials in transplantation and autoimmune diseases. FK506 modulates several cytokines and exerts hepatoprotection in acute models. This study was initiated to determine whether FK506 would be beneficial as an antifibrogenic agent. METHODS: Fibrosis was induced by carbon tetrachloride administration to rats. Half of those animals were treated with FK506. The rats were killed after 8 weeks of carbon tetrachloride treatment. The livers were evaluated by histology, Northern hybridizations, and collagen quantitation. Additionally, rat fibroblasts were incubated with and without FK506 and used for Northern hybridization analysis. RESULTS: Surprisingly, FK506 exacerbated hepatic inflammation and fibrosis. Increased hepatic collagen and higher messenger RNA levels of transforming growth factor beta 1 and collagens I, III, and IV were found in the FK506-treated group. Rat fibroblasts treated with FK506 expressed higher levels of collagens I and III, fibronectin, macrophage-colony stimulating factor, tissue inhibitor of metalloprotease, and transforming growth factor beta 1 messenger RNAs. CONCLUSIONS: These findings suggest that FK506 increases the expression of extracellular matrix genes and enhances fibrosis in the rat model. While further studies are needed to elucidate these profibrogenic mechanisms, caution is indicated for the unrestricted use of FK506 in patients subject to recurrent fibrogenic stimulation.

Animals↗
PubMed1999

Brain-derived neurotrophic factor infusion delays amygdala and perforant path kindling without affecting paired-pulse measures of neuronal inhibition in adult rats.

Kindling is an animal model of human temporal lobe epilepsy in which excitability in limbic structures is permanently enhanced by repeated stimulations. Kindling also increases the expression of nerve growth factor, brain-derived neurotrophic factor, and brain-derived neurotrophic factor receptor messenger RNAs in both the hippocampus and cerebral cortex and causes structural changes in the hippocampus including hilar hypertrophy. We have recently shown that intraventricular nerve growth factor infusion enhances the development of kindling, whereas blocking nerve growth factor activity retards amygdaloid kindling. Furthermore, we have shown that nerve growth factor protects against kindling-induced hilar hypertrophy. The physiological role of brain-derived neurotrophic factor in kindling is not as clear. Acute injection of brain-derived neurotrophic factor increases neuronal excitability and causes seizures, whereas chronic brain-derived neurotrophic factor infusion in rats slows hippocampal kindling. In agreement with the latter, we show here that intrahilar brain-derived neurotrophic factor infusion delays amygdala and perforant path kindling. In addition, we show that brain-derived neurotrophic factor, unlike nerve growth factor, does not protect against kindling-induced increases in hilar area. To test the hypothesis that brain-derived neurotrophic factor suppresses kindling by increasing inhibition above normal levels, we performed paired-pulse measures in the perforant path-dentate gyrus pathway. Brain-derived neurotrophic factor infused into the hippocampus had no effect on the stimulus intensity function (input/output curves); there was also no significant effect on paired-pulse inhibition. We then kindled the perforant path 10 days after the end of brain-derived neurotrophic factor treatment. Once again, kindling was retarded, showing that the brain-derived neurotrophic factor effect is long-lasting. These results indicate that prolonged in vivo infusion of brain-derived neurotrophic factor reduces, rather than increases, excitability without increasing inhibitory neuron function, at least as assessed by paired-pulse protocols. This effect may be mediated by long-lasting effects on brain-derived neurotrophic factor receptor regulation.

Amygdala↗
PubMed1988

An enhancer element in the short unique region of human cytomegalovirus regulates the production of a group of abundant immediate early transcripts.

A 275-bp sequence in the short unique region of human cytomegalovirus (HCMV) strain AD169 contains a series of five imperfect 18-bp repeats with homology to the SV40 and HCMV major immediate early enhancers. Plasmids containing these putative HCMV enhancer sequences linked to the human beta-globin gene were transiently transfected into HeLa cells, and the ability of the HCMV sequences to activate beta-globin transcription was assayed. The repeat-containing region stimulated transcription in a position- and orientation-independent manner, to approximately the same degree as that of the SV40 enhancer. A promoter located immediately 3' to the enhancer in the HCMV genome is active only in the presence of the enhancer sequences. Transcription from this HCMV promoter was analysed by a combination of S1 nuclease protection mapping and Northern blotting. At immediate early times postinfection, at least four extremely abundant differentially spliced mRNAs were detected; these RNAs constitute a previously unknown class of immediate early transcript.

Base Sequence↗
PubMed2006

Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue.

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed deltaNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (Cdelta8-Vero cells), a construct previously shown to be unable to complement DeltaNotI. In C-Vero cells but not Vero or Cdelta8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with deltaNotI (RUBrep/GFP-deltaNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5' and 3' cis-acting elements were also rescued in C-Vero cells. Interestingly, the Cdelta8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in Cdelta8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.

5' Untranslated Regions↗
PubMed2006

Role of the RNA2 3' non-translated region of Blackcurrant reversion nepovirus in translational regulation.

The 3' non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers (TEs). Blackcurrant reversion nepovirus (BRV) has a genome composed of two uncapped polyadenylated RNAs with very long 3' NTRs, nucleotide sequences of which are very conserved between different BRV isolates. In this work, we studied a role of the RNA2 3' NTR in translation, using mutagenesis of the firefly luciferase reporter mRNA, in protoplasts of Nicotiana benthamiana. The RNA2 3' NTR was found to contain a cap-independent TE (3' CITE), which must base pair with the 5' NTR to facilitate translation. The BRV 3' CITE and poly(A) tail provided a major contribution to translational efficiency, with less input from other 3' NTR parts. The BRV 3' CITE does not share similarity in nucleotide sequence and secondary structure with other viruses and thus represents a new class of 3' CITE.

3' Untranslated Regions↗
PubMed2002

Two distinct regions on the surface of an RNA-binding domain are crucial for RNase E function.

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.

Arginine↗
PubMed2002

Functional role of inflammatory cytokines and antiinflammatory molecules in seizures and epileptogenesis.

PURPOSE: We investigated the changes in the expression of proinflammatory cytokines and related molecules in the rodent hippocampus after the induction of limbic seizures. We then studied the effects of pharmacologic intervention on the interleukin (IL)-1 system on limbic seizures and the susceptibility to seizures of transgenic mice overexpressing the naturally occurring antagonist of IL-1 (IL-1Ra) in astrocytes. METHODS: Limbic seizures were induced in rodents by intrahippocampal injection of kainic acid or bicuculline methiodide or by electrical stimulation of the hippocampus causing status epilepticus (SE). Seizure activity was recorded by EEG analysis and behavioral observation according to Racine's scale. Cytokine expression in the hippocampus was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot quantification of the various messenger RNAs (mRNAs) and by immunocytochemistry. RESULTS: We found that limbic seizures rapidly and transiently enhanced IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha mRNA in the hippocampus with a peak effect at 6 h after SE. Immunoreactivity of the various cytokines was increased in glia. The increase of IL-1Ra was delayed because the peak effect was observed at 24 h after SE. Moreover, IL-1Ra was not produced in large excess, as during peripheral inflammation but in a molar ratio to IL-1beta of 1:1. Intrahippocampal injection of IL-1beta worsened seizure activity, whereas IL-1Ra was a powerful anticonvulsant in various models of limbic seizures. Transgenic mice overexpressing IL-1Ra in astrocytes were less sensitive to bicuculline-induced seizures. CONCLUSIONS: This study shows that limbic seizures in rodents rapidly and reversibly induce proinflammatory cytokines in glia and suggests that changes in the IL-1Ra/IL-1beta ratio in brain may represent an effective physiopathologic mechanism to control seizures.

Animals↗
PubMed2006

The role of NF-{kappa}B-1 and NF-{kappa}B-2-mediated resistance to apoptosis in lymphomas.

The NF-kappaB pathways have been implicated in tumorigenesis in several lymphoid malignancies, including non-Hodgkin's and Hodgkin's lymphomas. However, the antiapoptotic functions and the mechanism responsible for signaling through each NF-kappaB pathway remain to be elucidated. In the current study, lymphoma cell lines with constitutively active NF-kappaB were found to be resistant to inducers of the extrinsic and intrinsic apoptosis pathways. Resistance to cell death resulted from blocks early and late in the apoptosis cascade. Several NF-kappaB target genes were overexpressed in these cell lines, including Bcl-xL, Fas-associated death domain-like IL-1beta-converting enzyme inhibitor protein, cellular inhibitor of apoptosis, and X inhibitor of apoptosis. Inhibition of the canonical or noncanonical NF-kappaB pathways with small interfering RNAs or adenovirus expressing a stable form of inhibitor of NF-kappaB (IkappaB) enhanced sensitivity to apoptosis inducers and resulted in lower levels of Bcl-xL or Fas-associated death domain-like IL-1beta-converting enzyme inhibitor protein, cellular inhibitor of apoptosis, and X inhibitor of apoptosis. These findings demonstrate an important role of both NF-kappaB pathways in mediating resistance to apoptosis and distinctive antiapoptotic downstream target gene profiles responsible for this effect.

Adenoviridae↗
PubMed2005

Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation.

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

Amyloid beta-Protein Precursor↗
PubMed2026

abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas9 genome editing.

SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58 875 004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing. AVAILABILITY AND IMPLEMENTATION: The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.

Deep Learning↗
PubMed1979

Review: ethidium fluorescence assay. Part II. Enzymatic studies and DNA-protein interactions.

Almost all DNA and RNA metabolizing enzymes can be assayed rapidly and very sensitively by exploiting the enhanced fluorescence of ethidium intercalated into duplex DNA or RNA. Denatured DNA and natural RNAs contain duplex regions due to intramolecular hydrogen-bonding and can also be sensitively measured. Where the product is truly single-stranded (e.g. dTn) it can be assayed by adding the appropriate complementary strand (e.g. dAn or rAn). Some of the assays described provide information not readily obtained by other assay procedures. Among the enzymes readily assayed are DNA and RNA polymerases, terminal deoxynucleotidyl transferases, nucleases of all varieties (e.g. single-strand specific, endonucleases including for example AP endonucleases, exonucleases, RNase H, etc.), ligases, topoisomerases including gyrases, and indirectly enzymes such as proteases and superoxide dismutase. DNA binding proteins such as histones and helix destablizing proteins can also be quantitatively assayed.

DNA↗
PubMed2004

Influence of a fluorobenzene nucleobase analogue on the conformational flexibility of RNA studied by molecular dynamics simulations.

Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-beta-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-beta-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable 'base-paired' geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.

Adenine↗
PubMed2006

Activation of RegB endoribonuclease by S1 ribosomal protein requires an 11 nt conserved sequence.

The T4 RegB endoribonuclease cleaves specifically in the middle of the -GGAG- sequence, leading to inactivation and degradation of early phage mRNAs. In vitro, RegB activity is very weak but can be enhanced 10- to 100-fold by the Escherichia coli ribosomal protein S1. Not all RNAs carrying the GGAG motif are cleaved by RegB, suggesting that additional information is required to obtain a complete RegB target site. In this work, we find that in the presence of S1, the RegB target site is an 11 nt long single-stranded RNA carrying the 100% conserved GGA triplet at the 5' end and a degenerate, A-rich, consensus sequence immediately downstream. Our data support the notion that RegB alone recognizes only the trinucleotide GGA, which it cleaves very inefficiently, and that stimulation of RegB activity by S1 depends on the nucleotide immediately 3' to -GGA-.

Bacteriophage T4↗
PubMed2006

Identification of an RNA-silencing suppressor in the genome of Grapevine virus A.

Higher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.

Gene Expression↗
PubMed2000

Functional requirement for class I MHC in CNS development and plasticity.

Class I major histocompatibility complex (class I MHC) molecules, known to be important for immune responses to antigen, are expressed also by neurons that undergo activity-dependent, long-term structural and synaptic modifications. Here, we show that in mice genetically deficient for cell surface class I MHC or for a class I MHC receptor component, CD3zeta, refinement of connections between retina and central targets during development is incomplete. In the hippocampus of adult mutants, N-methyl-D-aspartate receptor-dependent long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is absent. Specific class I MHC messenger RNAs are expressed by distinct mosaics of neurons, reflecting a potential for diverse neuronal functions. These results demonstrate an important role for these molecules in the activity-dependent remodeling and plasticity of connections in the developing and mature mammalian central nervous system (CNS).

Animals↗
PubMed1992

Transfection-mediated recombination of influenza A virus.

Several mechanisms, including a high mutation rate and reassortment of genes, have been found to be responsible for the variability of influenza A viruses. RNA recombination would be another mechanism leading to genetic variation; however, recombination has only rarely been reported to occur in influenza viruses. During ribonucleoprotein transfection experiments designed to generate viable influenza viruses from in vitro-synthesized RNA, we discovered several viruses which must have originated from recombination events. The ribonucleoprotein transfection system may enhance the formation of viruses which result from jumping of the viral polymerase between RNAs or from ligation of different viral RNAs. Five different recombinant viruses are described. Two of these, REC1 and REC2, contain a neuraminidase (NA) gene whose defective polyadenylation signal has been repaired via intergenic recombination; 124 and 95 nucleotides have been added, respectively. Another virus, REC5, must have originated by multiple recombination events since it contains a mosaic gene with sequences derived from the NA gene of influenza A/WSN/33 virus and the matrix, polymerase protein PB1, and NA genes of influenza A/PR/8/34 virus.

Animals↗