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PubMed1994

Transmission of endogenous VL30 retrotransposons by helper cells used in gene therapy.

Retrovirus-derived vectors and packaging cell lines are basic components used for gene transfer in human gene therapy. To eliminate recombinational and transcriptional problems associated with retroviral vectors, synthetic retrotransposon VL30 vectors were devised. During experimentation with these new vectors, extensive cotransmission of endogenous VL30 retrotransposon sequences was observed, originating within helper cell lines used in gene therapy experiments. The RNA was efficiently packaged into helper virus and was transmitted to recipient human cells, where it was again expressed as RNA. Transmission occurred regardless of whether the vector was retrovirus-derived or VL30, or if no vector was used. Endogenous VL30 RNA was readily detected in unselected recipient cells after a single exposure to helper virus, demonstrating a high efficiency of transmission compared with a cotransmitted VL30 vector, which contained an extensive deletion of internal sequences. These studies indicated that VL30 retrotransposons were ubiquitously transmitted by murine helper cells. Furthermore, the data strongly suggested that improvements in gene transfer may be obtained, both by using nonmurine helper cells (to reduce competitive inhibition by endogenous VL30) and by using VL30-derived vectors with intact packaging sequences.

Animals↗
PubMed1993

Quantitation of tropoelastin mRNA and assessment of alternative splicing in human skin fibroblasts by reverse transcriptase-polymerase chain reaction.

We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the quantitative measurement of levels of tropoelastin mRNA in total RNA preparations from skin fibroblasts. This method facilitates the reproducible detection of low abundance tropoelastin mRNA in the range of 10-1000 copies per cell. The procedure is based on a competitive RT-PCR assay where a tropoelastin cDNA-derived internal RNA standard is cotranscribed and coamplified together with the sample derived-endogenous target mRNA. In addition, RT-PCR of several domains of tropoelastin mRNA, followed by DNA sequence analysis of asymmetric PCR products, revealed a previously unknown pattern of alternate exon usage at the 3' end of the tropoelastin gene in human skin fibroblasts.

Alternative Splicing↗
PubMed1999

Lack of reciprocal genomic imprinting of sense and antisense RNA of mouse insulin-like growth factor II receptor in the central nervous system.

Two models have been proposed to account for the molecular mechanism underlying genomic imprinting of the insulin-like growth factor II receptor gene (Igf2r): expression-competition and promoter DNA methylation. To examine which model best explains the regulation of Igf2r imprinting, we examined the allelic expression of endogenous Igf2r sense and antisense RNAs in mice. In peripheral tissues, Igf2r sense and antisense RNAs show a reciprocal pattern of imprinting and DNA methylation between the two parental alleles: the sense RNA is monoallelically expressed only from the maternal promoter which is unmethylated in region 1, and the antisense RNA is derived solely from the paternal promoter which is unmethylated in region 2. The paternal promoter of sense Igf2r and the maternal promoter of antisense Igf2r are hypermethylated and are transcriptionally suppressed. In CNS, the genomic imprinting of Igf2r sense and antisense RNAs is uncoupled: both parental promoters of Igf2r gene coding for sense RNA are unmethylated and are biallelically used for transcription. In contrast, antisense RNA of Igf2r is derived only from the paternal allele that is unmethylated in region 2, while the methylated maternal allele is silent. Uncoupling of genomic imprinting of Igf2r sense and antisense RNAs in CNS correlates with DNA methylation of the appropriate promoter region, thus favoring the model of DNA methylation over that of antisense as the chief regulator of Igf2r genomic imprinting.

Alleles↗
PubMed1974

Inhibition of uridine transport in cultured mammalian cells by theophylline.

Theophylline (theobromine, caffeine) reversibly inhibits the incorporation of labeled RNA precursors both in confluent 37 RC and in exponentially growing HeLa cells. As measured in 37 RC after 2 h labeling, 20 mM theophylline reduces the incorporation of [3H]UTP and [14C]uridine into acid-precipitable material to 5% and 9% of the control, respectively. This reduction is paralleled by a comparably lowered incorporation of the same precursors into the acid-soluble pool. The initial rate of incorporation into total cell material is similarly affected by theophylline, the inhibition being of a simple competitive type. Theophylline does not alter the turnover rate of pulse labeled RNA during actinomycin D chase nor does it preclude the utilization of the endogenous pool of nucleoside phosphates. Upto a concentration of 10 mM, it does not inhibit uridine kinase neither in 37 RC nor in HeLa cells. The mentioned inhibitory effects of theophylline cannot be mimicked by exogenously added cyclic AMP. All the data support the conclusion that theophylline inhibits the transport of uridine into the cell.

Biological Transport↗
PubMed1996

Adenovirus-mediated over-expression of interleukin-1 receptor antagonist reduces susceptibility to excitotoxic brain injury in perinatal rats.

In seven-day-old rats, intracerebral injection of N-methyl-D-aspartate transiently stimulates expression of Interleukin-1 beta messenger RNA. To evaluate the role of Interleukin-1 beta in the pathogenesis of excitotoxic injury, we sought to determine if Interleukin-1 receptor antagonist, an endogenous competitive inhibitor of Interleukin-1 beta, could attenuate N-methyl-D-aspartate-induced injury. To induce sustained over-expression of Interleukin-1 receptor antagonist in the brain, a recombinant adenovirus encoding Interleukin-1 receptor antagonist was administered by intracerebroventricular injection into three-day-old rats. Increased brain concentrations of Interleukin-1 receptor antagonist two to six days later were documented by assays of tissue homogenates and by immunocytochemistry. To evaluate the impact of Interleukin-1 receptor antagonist on N-methyl-D-aspartate neurotoxicity, three-day-old animals received intracerebroventricular injections of either adenovirus encoding Interleukin-1 receptor antagonist or a control adenovirus encoding beta-galactosidase, followed four days later by right intrastriatal injections of N-methyl-D-aspartate (10 nmol/0.5 microliter), a dose that typically elicits excitotoxic injury in the ipsilateral striatum and adjacent hippocampus, or saline. Animals were killed five days later, and brain damage was quantitated by measurement of bilateral cross-sectional areas of the striatum and anterior hippocampus. In three independent experiments, in N-methyl-D-aspartate-lesioned animals, both striatal and hippocampal injuries were reduced in animals that had been infected with adenovirus that encoded Interleukin-1 receptor antagonist, in comparison with littermates infected with the control adenovirus (right striatal volume loss ranged from 16 to 24%, compared with 54-65% volume loss in control). There was no striatal atrophy in adenovirus-infected saline-injected animals. These results provide strong support for the hypothesis that Interleukin-1 beta is a mediator of excitotoxic brain injury in perinatal rats.

Adenoviruses, Human↗
PubMed2000

Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing.

C to U editing of apolipoprotein B (apoB) mRNA involves the interaction of a multicomponent editing enzyme complex with a requisite RNA sequence embedded within an AU-rich context. This enzyme complex includes apobec-1, an RNA-specific cytidine deaminase, and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent the minimal core of the editing enzyme complex. The precise composition of the holo-enzyme, however, remains unknown. We have previously isolated an enriched fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with apobec-1, permits efficient C to U editing. Peptide sequencing of this most active fraction reveals the presence of ACF as well as GRY-RBP, an RNA-binding protein with approximately 50% homology to ACF. GRY-RBP was independently isolated from a two-hybrid screen of chicken intestinal cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA. Experiments using recombinant proteins demonstrate that GRY-RBP binds to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA editing. This competitive inhibition is rescued by addition of ACF, suggesting that GRY-RBP binds to and sequesters ACF. As further evidence of the role of GRY-RBP, rat hepatoma cells treated with an antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the nucleus of transfected cells and, in cotransfection experiments with apobec-1, each appears to colocalize in a predominantly nuclear distribution. Taken together, the results indicate that GRY-RBP is a member of the ACF gene family that may function to modulate C to U RNA editing through binding either to ACF or to apobec-1 or, alternatively, to the target RNA itself.

APOBEC-1 Deaminase↗
PubMed2004

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

Acetylcysteine↗
PubMed1977

Endogenous RD-114 virus genome expression in malignant tissues of domestic cats.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.

Animals↗
PubMed1993

Partial purification and characterization of foxtail mosaic potexvirus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase (RdRp) of foxtail mosaic virus (FMV) was partially purified from infected leaves of Chenopodium quinoa. The membrane fraction of crude plant extracts contained most of the FMV RdRp activity. Additional purification was obtained by solubilization of the RdRp using KCl and dodecyl-sucrose and by centrifugation through a glycerol gradient. The RNA template endogenous to RdRp preparations could be removed using micrococcal nuclease but the resulting fraction was unable to copy added template purified from FMV virions. However, supplementation of fractions containing RdRp activity with FMV RNA resulted in a significant decrease in the level of RNA synthesis. This effect was specific to potexviral RNAs since a similar interference was also observed with clover yellow mosaic virus RNA but not with brome mosaic virus RNA or yeast RNA. RNA transcripts corresponding to various regions of the FMV genome were tested for their ability to inhibit RNA synthesis on endogenous template. The simultaneous presence of both 5' and 3' terminal regions of the viral genome was necessary to interfere with RNA synthesis suggesting that this inhibition resulted from competition for the binding of component(s) of the RdRp complex.

Base Sequence↗
PubMed1985

Growing Xenopus oocytes have spare translational capacity.

Previous studies have shown that exogenous mRNAs injected into full-grown (stage 6) Xenopus oocytes are translated only at the expense of endogenous messages; translational capacity is limited. In this report, we demonstrate that injection of globin mRNA into small, stage 4 oocytes results in an increase in total protein synthesis without a concomitant decrease in the translation of endogenous mRNAs. The absence of competition with endogenous messages in stage 4 oocytes, injected with globin mRNA, compared with stage 6 oocytes, was not due to differential turnover of the injected mRNA. Hybridization of RNA from mRNA-injected oocytes at both stages revealed that similar amounts of globin mRNA were present. These results are interpreted to mean that protein synthesis in growing oocytes is limited by the availability of mRNA and not components of the translational machinery. This conclusion, however, does not apply to all mRNA classes. The capacity of stage 4 oocytes to translate a mRNA (zein) on membrane-bound polysomes is as restricted as reported previously for stage 6 oocytes. This result suggests that putative membrane binding sites or rough endoplasmic reticulum content are limited in oocytes at both stages. The possible role of association of injected mRNA with endogenous proteins that prevent translation is discussed.

Animals↗
PubMed1994

Interaction between tumour necrosis factor alpha ribozyme and cellular proteins. Involvement in ribozyme stability and activity.

Ribozymes are RNA molecules that cleave other RNA molecules. Thus, ribozymes offer a new way of inhibiting expression of specific genes whose nucleotide sequences are known. Intracellular stability of ribozymes is an important factor for their efficacy. We previously showed that hammerhead ribozyme directed against mRNA of tumour necrosis factor alpha (TNF alpha) slowly acquires resistance to degradation in cultured human cells. In order to explain this resistance, we now report on endogenous cellular protein(s) that bind to TNF alpha-ribozyme (TNF alpha-Rz) in solution to form stable complexes during native gel electrophoresis. Suppression of the effects of ribonucleases in the cytoplasmic extracts allowed approximately 80% of the input ribozyme RNA to be recovered in the form of complexes, indicating that complex formation protected the ribozyme from degradation. Treatment of the ribozyme-protein complexes with proteinase K prior to electrophoresis led to the recovery of full-length ribozyme. Interestingly, ribozyme-protein complexes retained cleavage activity, suggesting that the binding is in reversible equilibrium. Analysis of protein cytoplasmic extracts for binding to sub-fragments of TNF alpha-Rz demonstrated that protein binds to a conformational epitope formed by an interaction between the 5' end of TNF alpha-Rz and its catalytic domain. Competition of the ribozyme-protein binding with a ribozyme construct containing DNA instead of RNA at the 5' end, indicated that the ribose phosphate backbone of the 5' end is required for strong binding. The protein responsible for the formation of the complex with low electrophoretic mobility was found to be specific for the TNF alpha-Rz, since ribozyme for HIV-1 integrase gene (Int-Rz), or for human interleukin-2 (IL2-Rz) did not compete significantly with the TNF alpha-Rz binding. Covalent linkage of the IL2-Rz to the 3' end of TNF alpha-Rz, or to the proposed RNA protein binding site conferred protein binding and enhanced the stability and activity of the chimeric molecules. The RNA epitope identified in this study, through its endogenous protein binding, may serve as an oligonucleotide cassette for enhancing the in vivo stability and activity of other RNA molecules in general. This RNA epitope will also be useful in the study of RNA-protein interactions.

Base Sequence↗
PubMed1977

Biochemical properties of endogenous rat C-type viruses.

The cellular restriction to persistence of inducible C-type viruses of at least two rat strains can be overcome in actively in vitro replicating and/or spontaneously transforming rat embryo cells. Both, SD-RaLV and W-RaLV particles are morphologically and biochemically related to known oncorna viruses: They are of C-type, have an identical buoyant density of 1.15 g/cm3 in sucrose gradients and contain high-molecular-weight RNA. The RaLV-associated DNA polymerase exhibit both exogenous (polynucleotide-templated) and endogenous (viral RNA-templated) reactivity. The enzyme strongly preferred Mn++ over Mg++ ions (Mg++/Mn++ ratio of 0.04 to 0.07), with maximum reactivity at 0.1 mM MnCl2 concentration. The molecular hybridization experiments revealed the endogenous nature of the SD-RaLV and the W-RaLV. From the competition hybridization assay the number of proviral sequences could be calculated to be 84 per haploid cell genome. The role of endogenous rat viruses in the "spontaneous" in vitro transformation is discussed.

Animals↗
PubMed2000

Lack of clinical evidence for involvement of hepatitis C virus interferon-alpha sensitivity-determining region variability in RNA-dependent protein kinase-mediated cellular antiviral responses.

The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system. Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR. The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver. ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material. As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN-alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique. Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression. The data suggest that at least two prominent PKR-mediated cellular responses might be largely unaffected by HCV ISDR variability.

Adolescent↗
PubMed1979

Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.

Animals↗
PubMed1983

Hamster endogenous retrovirus (HaER)--distinct properties of structural proteins and DNA polymerase.

The structural proteins as well as some features of the RNA-dependent DNA polymerase of the hamster endogenous retrovirus (HaER) were examined. The polypeptide pattern of this virus is substantially different from that of other known retroviruses in containing major polypeptides with molecular weights of 68,000, 59,000, 27,000 and 24,000 daltons. Double antibody competitive radioimmunoassays showed that the HaER particles do not share any detectable antigenic relatedness with the murine viruses' p30, but manifest a considerable relatedness with the feline leukemia virus p27 and a slight cross-reactivity with the rat virus major protein. The RNA-dependent DNA polymerase of HaER virus has a molecular size of approximately 73,000 daltons and in contrast to other mammalian retroviruses shows no significant preference for Mn2+ over Mg2+. Apart from the lack of antigenic relatedness between the HaER virus proteins and the p30 protein of murine viruses, there is also no antigenic relatedness between HaER and murine viruses insofar as their DNA polymerase is concerned.

Animals↗
PubMed2006

TbVps34, the trypanosome orthologue of Vps34, is required for Golgi complex segregation.

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division.

Animals↗
PubMed2006

Sphingosine regulates the transcription of CYP17 by binding to steroidogenic factor-1.

Steroidogenic factor (SF1, Ad4BP, NR5A1) is a nuclear receptor that is essential for steroid hormone biosynthesis and endocrine development. Recent crystallographic studies have found that phospholipids are ligands for SF1. In the present study, our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using tandem mass spectrometry, we show that in H295R adrenocortical cells, SF1 is bound to sphingosine (SPH) and lyso-sphingomyelin (lysoSM) under basal conditions and that cAMP stimulation decreases the amount of SPH and lysoSM bound to the receptor. Silencing both acid and neutral ceramidases using small interfering RNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. SPH antagonized the ability of cAMP and the coactivator steroid receptor coactivator-1 to increase CYP17 reporter gene activity. These studies demonstrate that SPH is a bonafide endogenous ligand for SF1 and a negative regulator of CYP17 gene expression.

Binding Sites↗