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At least 631 records · Page 35Linked to original sources
PubMed2001

Genomic analysis of differentially expressed genes in liver and biliary epithelial cells of patients with primary biliary cirrhosis.

The characterization of differentially expressed genes provides a powerful tool for identifying molecules that may be involved in the pathogenesis of disease. We have used two independent techniques to identify overexpressed transcripts in bile duct cells and in liver from patients with primary biliary cirrhosis (PBC). In the first method, we used suppressive subtractive hybridization to compare mRNA from isolated PBC bile duct epithelial cells (BECs) to normal BECs and identified 71 clones as transcribed at higher levels in PBC-BECs. Amongst these clones, 62/71 had matches in a non-redundant nucleotide database and 9/71 had matches in an EST database. Of the 62 clones, 51/62 include a complexity of genes involved in cell proliferation, signal transduction, transcription regulation, RNA processing, carbohydrate metabolism and hypothetical/unknown proteins; 4/62 were identified as interstitial collagenase and collagenase precursors, 4/62 as ribosomal proteins, 3/62 as mitochondrial DNA. The mitochondrial cDNA sequences included cytochrome c oxidase, Wnt-13, and the pHL gene, a c-myc oncogene containing coxIII sequence. In the second method, we constructed cDNA libraries from three different PBC livers and sequenced a total of 12,324 independent clones. These 12,324 clones underwent virtual subtraction with 2,814,148 independent clones from Incyte LifeSeq libraries. Twenty one sequences were identified as unique to PBC liver. Collectively, these approaches identified a number of genes involved in signalling, RNA processing, mitochondrial function, inflammation, and fibrosis. Interestingly, both Wnt-13 and Notch transcripts are overexpressed in PBC liver. Further studies are needed to focus on the significance of these genes during the natural history of disease.

Bile Ducts↗
PubMed1996

Sex-lethal interacts with splicing factors in vitro and in vivo.

The Drosophila sex determination gene Sex-lethal controls its own expression and the expression of downstream target genes such as transformer by regulating RNA splicing. Genetic and molecular studies have established that Sxl requires the product of another gene, snf, to autoregulate the splicing of its own transcripts. snf has recently been shown to encode a Drosophila U1 and U2 small nuclear ribonucleoprotein particle protein. In the work reported here, we demonstrate that the Sxl and Snf proteins can interact directly in vitro and that these two proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with the Sxl antibody. Unlike bulk Snf protein, which sediments slowly in sucrose gradients, the Snf protein associated with Sxl is in a large, rapidly sedimenting complex. Detailed characterization of the Sxl-Snf complexes from cross-linked extracts indicates that these complexes contain additional small nuclear ribonucleoprotein particle proteins and the U1 and U2 small nuclear RNAs. Finally, consistent with the RNase sensitivity of the Sxl-Snf complexes, Sxl transcripts can also be immunoprecipitated by Sxl antibodies. On the basis of the physical interactions between Sxl and Snf, we present a model for Sxl splicing regulation. This model helps explain how the Sxl protein is able to promote the sex-specific splicing of Sxl transcripts, utilizing target sequences that are distant from the regulated splice sites.

Animals↗
PubMed2000

Down-regulation of the endoplasmic reticulum chaperone GRP78/BiP by vomitoxin (Deoxynivalenol).

The mechanisms by which trichothecene mycotoxins cause immunological effects in leukocytes such as cytokine up-regulation, aberrant IgA production, or apoptotic cell death are not fully understood. In the present study, mRNA differential display analysis was used to evaluate changes in gene expression induced by the trichothecene vomitoxin (VT or deoxynivalenol) in a T-cell model, the murine EL-4 thymoma, that was stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION). Ten differentially expressed fragments of cDNA were isolated and sequenced and three of these were identified as the known genes GRP78/BiP, P58(IPK), and RAD17. Most notably, expression of GRP78/BiP (a 78-kDa glucose-regulated protein), a stress-response gene induced by agents or conditions that adversely affect endoplasmic reticulum (ER) function, was found to decrease in VT-exposed cells. Competitive RT-PCR analysis revealed that 250 ng/ml VT decreased GRP78/BiP mRNA expression in both unstimulated and PMA/ION-stimulated EL-4 cells at 6 and 24 h after VT treatment. Western blotting confirmed that VT (50 to 1000 ng/ml) also significantly diminished GRP/BiP protein levels in a dose-response manner in PMA/ION-stimulated cells. GRP78/BiP has been shown to play a role in regulation of protein folding and secretion, and to protect cells from apoptosis. When PMA/ION-stimulated cells were incubated with 50 to 1000 ng/ml VT for 24 h, 200-bp DNA laddering, a hallmark of apoptosis, increased in a dose-dependent manner. In addition to GRP78, mRNA expression of the cochaperone P58(IPK), which is the 58-kDa cellular inhibitor of the double-stranded RNA-regulated protein kinase (PKR), was also shown to be suppressed by VT-treatment. GRP78 and P58(IPK) are critical for maintenance of cell homeostasis and prevention of apoptosis. The down-regulation of these molecular chaperones by VT represent a novel observation and has the potential to impact immune function at multiple levels.

Animals↗
PubMed2003

Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPase(old-35), a Type I interferon inducible early response gene.

An overlapping pathway screening (OPS) approach designed to identify and clone genes displaying parallel expression profiles as a function of induction of terminal differentiation and cellular senescence in human cells identified a novel gene old-35. Sequence and functional analysis indicates that old-35 encodes human polynucleotide phosphorylase, hPNPase(old-35). Polynucleotide phosphorylases comprise a family of phosphate dependent 3'-5' RNA exonucleases implicated in RNA regulation. Treatment of HO-1 human melanoma and additional diverse normal and tumor-derived human cell types with Type I interferon (IFN), IFN-beta or IFN-alpha, induces hPNPase(old-35) expression. To provide insights into the regulation of hPNPase(old-35), we cloned and analyzed the promoter region of this gene. These studies demonstrate that IFN-beta controls hPNPase(old-35) expression by transcriptional modulation rather than by altering mRNA stability. Transcriptional activation of hPNPase(old-35) by IFN-beta is primarily mediated by the interferon stimulatory response element (ISRE) present in its promoter. Analysis of hPNPase(old-35) expression in cell lines defective in various IFN signaling molecules confirms that hPNPase(old-35) expression is dependent upon the Janus activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Furthermore, gel shift analyses document that hPNPase(old-35) is a direct target of the interferon stimulated gene factor 3 (ISGF3) complex. The hPNPase(old-35) gene spans approximately 54 kb of genomic DNA and is distributed on 28 exons and 27 introns. hPNPase(old-35) maps to 2p15-2p16.1, a region implicated in hereditary nonpolyposis colorectal cancer, Carney complex, Doyne's honeycomb retinal dystrophy and several other diseases. To provide insights into PNPase function in vivo, we have also cloned the mouse PNPase(old-35) cDNA, mPNPase(old-35). Induction of hPNPase(old-35) by IFN treatment as well as during differentiation and senescence suggest that this gene may play a significant role in regulating cellular growth and that overlapping gene expression changes, also induced by IFN, may contribute to these important physiological processes.

5' Untranslated Regions↗
PubMed1991

The lentivirus regulatory proteins REV and REX are site specific RNA binding proteins.

These studies demonstrate that HIV-1 REV and HTLV-1 REX are site specific RNA binding proteins. In addition, the REV protein studies indicate a protein domain essential for biological function that is not involved in RRE binding. Lentiviruses are unique among retroviruses in providing gene products to switch expression from early to late genes. Rather than transcriptional control, this is accomplished by regulating RNA transport. A further understanding of these lentiviral genes may identify means of inhibiting viruses responsible for insidious human disease.

Amino Acid Sequence↗
PubMed1990

Cell-specific expression of kidney androgen-regulated protein messenger RNA is under multihormonal control.

Kidney androgen-regulated protein (KAP) gene expression is under androgenic control in the epithelial cells of the proximal convoluted tubule in the mouse kidney. In Tfm/Y androgen receptor-deficient mice, KAP mRNA was detected by in situ hybridization in a subpopulation of these cells only in the S3 segment of the proximal tubules in the outer medulla. Treatment of Tfm/Y animals with testosterone caused a partial induction of KAP mRNA levels, while dihydrotestosterone had no effect. These data suggested that the androgen receptor-independent induction of KAP gene expression in these animals was mediated by an estrogenic metabolite of testosterone, since dihydrotestosterone cannot be aromatized to an estrogenic form. Estrogen treatment of Tfm/Y mice caused an increase in KAP gene expression similar to that observed with testosterone. However, ovariectomy of normal female mice did not eliminate KAP gene expression in the S3 cells and, in fact, resulted in a slight increase. Adrenalectomy in combination with castration had no effect on KAP mRNA levels in S3 cells. However, hypophysectomy alone completely eliminated this cell-specific component of KAP gene expression. These results indicate that KAP gene expression is subject to cell-specific regulation in different segments of the proximal tubule and that this regulation is mediated by hormones of both gonadal and pituitary origin.

Androgens↗
PubMed1991

Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro.

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins. The mechanisms by which these proteins regulate RNA processing has not been characterized. In this paper we show that tra-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of dsx pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing.

Animals↗
PubMed1994

Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells.

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.

Alternative Splicing↗
PubMed1999

The widely conserved Era G-protein contains an RNA-binding domain required for Era function in vivo.

Era is a small G-protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G-proteins and is required for Era function in vivo. The C-terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA-binding domain. Although both contain the consensus VIGxxGxxI RNA-binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher-order RNA-protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L-66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.

Amino Acid Motifs↗
PubMed1989

HTLV-1 rex and HIV-1 rev act through similar mechanisms to relieve suppression of unspliced RNA expression.

Human retroviruses, human T cell leukemia viruses (HTLV) and human immunodeficiency viruses (HIV), express two classes of mRNAs; fully spliced mRNA in the early phase and intron-containing mRNA in a later phase. The expressions of HTLV-1 rex and HIV rev by early mRNAs are essential for the later phase of expression of intron-containing gag and env mRNAs. Each two cis-acting sequences seem to be involved in these regulations: HTLV-1 rex depends on a splice donor (SD) and a responsible element (RXE) at the 3' end, whereas HIV rev depends on a specific repressive sequence (CRS) and a responsible element (RRE) in the intron, but does not require an SD. For analyses of these cis-acting sequences, we inserted an HIV element RRE into an HTLV-1 construct and tested the responses to HTLV-1 rex and HIV rev regulations. The results indicated that both rex and rev could regulate RNA expression of these chimeric constructs responding to an HIV RRE. A repressive element (CRS) was dispensable, and the intronic or exonic location of RRE was not important. These observations suggest that rex and rev could be functionally equivalent to induce cytoplasmic expression of unspliced RNA which expression is suppressed either by an SD or CRS depending on the construction.

Cell Line↗
PubMed1991

Inhibition of c-erbA mRNA splicing by a naturally occurring antisense RNA.

The rat erbA alpha locus encodes two overlapping mRNAs, alpha 1 and alpha 2, which are identical except for their most 3' exons. alpha 1 mRNA encodes a thyroid hormone receptor, while alpha 2 encodes an altered ligand binding domain of unknown function. Previous studies have shown that the ratio of alpha 1 to alpha 2 is highest in cells expressing a high level of a third RNA, Rev-ErbA alpha mRNA, which is transcribed in the opposite direction and is complementary to alpha 2 but not alpha 1 mRNA. It was hypothesized that base pairing with Rev-ErbA alpha blocks splicing of alpha 2 mRNA, thereby favoring formation of the non-overlapping alpha 1. To test this model, a system was developed in which alpha 2 pre-mRNAs were accurately spliced in vitro. Splicing was inhibited by the addition of a 5-fold excess of antisense RNAs containing the 3' end of Rev-ErbA alpha mRNA. Both an antisense RNA extending across the 3' splice site and a shorter RNA complementary only to exon sequences efficiently blocked splicing. However, splicing was only inhibited by complementary RNAs. These observations are consistent with a mechanism in which base pairing with a complementary RNA regulates alternative processing of alpha 1 and alpha 2 mRNAs.

Animals↗
PubMed2006

MicroRNA-targeted and small interfering RNA-mediated mRNA degradation is regulated by argonaute, dicer, and RNA-dependent RNA polymerase in Arabidopsis.

ARGONAUTE1 (AGO1) of Arabidopsis thaliana mediates the cleavage of microRNA (miRNA)-targeted mRNAs, and it has also been implicated in the posttranscriptional silencing of transgenes and the maintenance of chromatin structure. Mutations in AGO1 severely disrupt plant development, indicating that miRNA function and possibly other aspects of RNA interference are essential for maintaining normal patterns of gene expression. Using microarrays, we found that 1 to 6% of genes display significant expression changes in several alleles of ago1 at multiple developmental stages, with the majority showing higher levels. Several classes of known miRNA targets increased markedly in ago1, whereas others showed little or no change. Cleavage of mRNAs within miRNA-homologous sites was reduced but not abolished in an ago1 -null background, indicating that redundant slicer activity exists in Arabidopsis. Small interfering RNAs and larger 30- to 60-nucleotide RNA fragments corresponding to highly upregulated miRNA target genes accumulated in wild-type plants but not in ago1, the RNA-dependent RNA polymerase mutants rdr2 and rdr6, or the Dicer-like mutants dcl1 and dcl3. Both sense and antisense RNAs corresponding to these miRNA targets accumulated in the ago1 and dcl1 backgrounds. These results indicate that a subset of endogenous mRNA targets of RNA interference may be regulated through a mechanism of second-strand RNA synthesis and degradation initiated by or in addition to miRNA-mediated cleavage.

Animals↗
PubMed1996

Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

Animals↗
PubMed2006

Properties of novel antisense oligonucleotides containing 2'-O-modified adenosine with a photo-reactive group.

In order to enhance the efficiency of antisense molecules for target RNA regulation, a novel photo-reactive antisense oligonucleotide was developed. We designed and synthesized a photo-reactive antisense oligonucleotide containing an adenosine in which 2'-OH was modified with 4,5',8-trimethylpsoralen (Ps) via an ethoxymethylene linkage (2'-Ps-eom). We evaluated the photo-cross-linking efficiency and sequence specificity toward complementary RNA (match-RNA). 2'-Ps-eom was selectively photo-cross-linked to the match-RNA. The photo-cross-linking efficiency was about 75% upon UVA-irradiation (365 nm) for 10 min. Previously, we reported oligonucleotides that had an adenosine anchoring Ps at 2'-O-position via a methylene linkage (2'-Ps-met). The photo-cross-linking efficiency of 2'-Ps-met and match-RNA was about 35% upon UVA-irradiation for 120 min. The photo-cross-linking efficiency of 2'-Ps-eom was dramatically enhanced in comparison with the one of 2'-Ps-met.

Adenosine↗
PubMed2000

Rapid induction of nuclear transcripts and inhibition of intron decay in response to the polymerase II inhibitor DRB.

The transcriptional inhibitor 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) is an adenosine analog that has been shown to cause premature transcriptional termination and thus has been a useful tool to identify factors important for transcriptional elongation. Here, we establish an efficient system for studying DRB-sensitive steps of transcriptional elongation. In addition, we establish two novel effects of DRB not previously reported: intron stabilization and the induction of long transcripts by a mechanism other than premature termination. We found that DRB had a biphasic effect on T-cell receptor-beta (TCRbeta) transcripts driven by a tetracycline (tet)-responsive promoter in transfected HeLa cells. In the first phase, DRB caused a rapid decrease (within five minutes) of pre-mRNA and its spliced intron (IVS1(Cbeta1)), consistent with the known ability of DRB to inhibit transcription. In the second phase (which began ten minutes to two hours after treatment, depending on the dose), DRB dramatically increased the levels of IVS1(Cbeta1)-containing transcripts by a mechanism requiring de novo RNA synthesis. DRB induced the appearance of short 0.4 to 0.8 kb TCRbeta transcripts in vivo, indicating DRB enhances premature transcriptional termination. A approximately 475 nt prematurely terminated transcript (PT) was characterized that terminated at an internal poly(A) tract in the intron IVS1(Cbeta1). We identified three other effects of DRB. First, we observed that DRB induced the appearance of heterodisperse TCRbeta transcripts that were too long ( approximately 1 kb to >8 kb) to result from the type of premature termination events previously described. Their production was not promoter-specific, as we found that long transcripts were induced by DRB from both the tet-responsive and beta-actin promoters. Second, DRB upregulated full-length normal-sized c-myc mRNA, which provided further evidence that DRB has effects besides regulation of premature termination. Third, DRB stabilized lariat forms of the intron IVS1(Cbeta1), indicating that DRB exerts post-transcriptional actions. We propose that our model system will be useful for elucidating the factors that regulate RNA decay and transcriptional elongation in vivo.

Cell Nucleus↗
PubMed1995

A model for the sequence-dependent DNA binding of 4',6-diamidino-2-phenylindole (DAPI).

A study on the sequence dependent DNA binding mode of DAPI has been carried out on pUC8 and the beta gal promoter region by restriction endonuclease and DNAase I protection experiments. A molecular model depicting drug interaction at the level of selected palyndromes has also been constructed that confirms the A-T sequence specificity of the compound. Experimental data indicate that the binding sites for RNA polymerase and cyclic AMP receptor protein (CRP) in the beta gal gene are privileged locales for DAPI interaction, a feature that explains impairment of transcription at this level. From a stereochemical view point, DAPI binding to DNA minor groove, while being incompatible with promoter unwinding in the open complex, may also disturb optimal contacts with proteins regulating RNA polymerase activity.

Base Composition↗
PubMed2004

Small stable RNA level depends on the physiological state of the cell.

The level of 5S rRNA and tRNAi(Met)1 synthesized by RNA polymerase III was investigated in human epidermoid carcinoma cells A431 at different physiological states: low and high proliferation and apoptosis. The real-time RT-PCR method using SYBR Green I was applied to measure certain RNA species in total cellular RNA. The share of 5S rRNA was practically the same in slowly and actively proliferating A431 cells, but increased about 2.5-fold in apoptotic cells. The share of initiator tRNAi(Met)1 in actively proliferating and apoptotic cells was 1.5-2.0 times higher than in slowly proliferating cells. Our results suggest a possible existence of special mechanisms regulating RNA polymerase III-directed transcription from different type promoters in accordance with the physiological state of the cell.

Apoptosis↗
PubMed2003

CotB is essential for complete activation of green light-induced genes during complementary chromatic adaptation in Fremyella diplosiphon.

The dramatic modifications of photosynthetic light harvesting antennae called phycobilisomes that occur during complementary chromatic adaptation in cyanobacteria are controlled by two separate photosensory systems. The first system involves the signal transduction components RcaE, RcaF and RcaC, which appear to make up a complex multistep phosphorelay. This system controls the light responsive expression of the cpcB2A2H2I2D2, cpeBA and cpeCDE operons, which encode phycobilisome proteins. The second system, which is not yet characterized, acts in concert with the first but only regulates the light responses of cpeBA and cpeCDE. We have generated and characterized a new mutant class, named the Tan mutants. In at least one member of this class, light-regulated RNA accumulation patterns are altered for cpeBA and cpeCDE, but not for cpcB2A2H2I2D2. Thus this mutant contains a lesion that may impair the operation of the second system. We demonstrate that several Tan mutants are the result of improper expression of the gene cotB. CotB has limited similarity to lyase class proteins, particularly those related to NblB, which is required for degradation of phycobilisomes in other cyanobacteria. Possible roles of CotB in the biogenesis of phycobilisomes are discussed.

Adaptation, Physiological↗