Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies indicate that a peroxisome proliferator-activated receptor, PPAR gamma, functions as an important adipocyte determination factor. In contrast, tumor necrosis factor-alpha (TNF alpha) inhibits adipogenesis, causes dedifferentiation of mature adipocytes, and reduces the expression of several adipocyte-specific genes. Here, we report that treatment of 3T3-L1 adipocytes with TNF alpha resulted in a time- and concentration-dependent decrease in PPAR gamma mRNA expression to the level detected in preadipocytes. PPAR gamma mRNA levels were reduced by 95% with 3 nM TNF alpha treatment for 24 h. Half-maximal effects were seen after 3 h treatment with 3 nM TNF alpha or with 50 pM TNF alpha (24-h exposure). Parallel reductions in PPAR gamma protein levels were also observed after treatment of 3T3-L1 adipocytes with TNF alpha. Using a ribonuclease protection assay, both alternatively spliced PPAR gamma isoforms (gamma 1 and gamma 2) were shown to be negatively regulated by TNF alpha. The down-regulation of PPAR gamma by TNF-alpha preceded the diminution in expression of other adipocyte-specific genes including CCAAT/enhancer binding protein and adipocyte fatty acid-binding protein (aP2). The effect of TNF alpha was specific for the gamma-isoform of PPARs, since the expression of PPAR delta mRNA was not affected by treatment with TNF alpha. Low level constitutive expression of PPAR gamma in 3T3-L1 adipocytes (at levels approximately 2- to 3-fold higher than in preadipocytes) partially blocked the inhibitory effect of TNF alpha on aP2 and adipsin expression. These findings support the following conclusions: 1) PPAR gamma expression is necessary for the maintenance of the adipocyte phenotype. 2) PPAR gamma, but not PPAR delta, expression is sufficient to attenuate TNF alpha-mediated effects on adipocyte phenotype. 3) Reduced PPAR gamma gene expression is likely to represent an important component of the mechanism by which TNF alpha exerts its antiadipogenic effects.
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PMID:Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha. 892 70

Congestive heart failure leads to skeletal muscle abnormalities, one of which is a prolongation of sarcoplasmic reticulum Ca2+ flux. The purpose of this study was to determine whether skeletal muscle of spontaneous hypertensive and heart failure rats have alterations in the expression of the sarcoplasmic (or endoplasmic) reticulum Ca(2+)-ATPase (SERCA) gene. Northern analysis revealed that SERCA1, the predominant skeletal muscle isoform, was decreased by 45%, 43%, and 58% in the tibialis anterior, plantaris, and diaphragm muscles, respectively. Ribonuclease protection assay showed that the decrease was due to the adult isoform, SERCA1a, with minor changes in the alternatively spliced neonatal isoform, SERCA1b. There was no change in SERCA1 mRNA levels in gastrocnemius muscles. No change was found in SERCA2a (cardiac/slow skeletal isoform) mRNA or protein levels or in SERCA2b (smooth muscle isoform), dihydropyridine receptor, or alpha-actin mRNA levels in diaphragm muscle. Northern blot and ribonuclease protection assays showed that SERCA2a decreased 61% in the heart while the alternatively spliced isoform, SERCA2b, decreased 27%. Western analysis of the tibialis anterior, diaphragm, and gastrocnemius muscles showed a decrease in SERCA1 protein levels by 46%, 64%, and 42%, respectively, whereas sarcoplasmic reticulum Ca(2+)-ATPase activity, a functional correlate of SERCA expression, was decreased by 38%, 38%, and 40% in the same muscles, SERCA2 protein expression decreased by 36% in the failing heart. Decreases in both mRNA and protein suggest pretranslational control of SERCA1 expression, whereas the lack of decreased SERCA1 mRNA in gastrocnemius muscle suggests translational regulation. The decreased SERCA1 protein expression in all muscles studied probably contributes to contractile abnormalities related to excitation-contraction coupling function in heart failure.
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PMID:Skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase gene expression in congestive heart failure. 935 44

Localization of tenascin-C in vivo and cell culture experiments in vitro have provided evidence for stromal production of tenascin-C in malignant tumors of a variety of organs. Here we raised the question of whether the mesenchymal stroma in the case of endometrial adenocarcinoma is the unique source of tenascin-C. Therefore, the expression of tenascin-C mRNA by human endometrial adenocarcinoma cells and endometrial stroma cells was investigated. Several preparations of endometrial stroma cells produced tenascin-C mRNA. Using a serum-free defined cell culture medium, production of tenascin-C mRNA could be increased by adding either serum or 20 ng TGF-beta/mL to the cell culture medium. Reverse transcriptase polymerase chain reaction analysis revealed that five out of six endometrial adenocarcinoma cell lines produced tenascin-C mRNA. Northern blot experiments and ribonuclease protection assays provided evidence that the number of copies of tenascin-C mRNA was small. Analysis of expressed splice variants by reverse transcriptase polymerase chain reaction analysis revealed the abundance of one major splice variant that lacked all potential alternatively spliced fibronectin type-III-like repeats. Regarding larger splice variants, all fragment sizes that could theoretically originate from seven alternatively spliced fibronectin type-III-like repeats were observed. Evaluating relative signal intensities, the splice variants containing a single fibronectin type-III-like repeat and the variant possessing all but one alternatively spliced repeats were most frequent. In summary, evidence is provided that tenascin-C can originate from both tissue compartments of the human endometrium stroma and (tumor) epithelium. Splice variant analysis revealed a high number of splice variants and a relative high proportion of variants that have so far been regarded as minor constituents of expressed tenascin-C.
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PMID:Expression of tenascin-C by human endometrial adenocarcinoma and stroma cells: heterogeneity of splice variants and induction by TGF-beta. 959 65

Type XI collagen, a member of the group of fibrillar collagens, plays a regulatory role in the formation of the collagen fibril network in cartilage and consequently plays a pivotal role in the formation of the endochondral skeleton. The mechanism by which type XI collagen limits fibril growth appears to involve the large noncollagenous amino terminal domain. Complex alternative splicing occurs within this domain in two of the three constituent subunits, alpha1(XI) and alpha2(XI). In the alpha1(XI) chain, three alternatively spliced exons encoding one very basic and two very acidic peptides generate six spliceforms and protein isoforms. In order to better understand the significance of this alternative splicing, we have examined fetal rat cartilage to determine: (a) the relationship between alternative splicing and chondrogenesis in limb bud micromass culture; (b) the relative levels of expression of each of the splice-forms by ribonuclease protection; and (c) the distribution of splice-forms and protein isoforms by in situ hybridization and immunohistochemistry. The results indicate that the pattern of alternative splicing of the alpha1(XI) chain is tightly linked to chondrogenesis. The two most abundant spliceforms in fetal rib cartilage are v(o), lacking all three exons, and v1b, containing the exon encoding the basic peptide. While most of the spliceforms show a general distribution in nasal, Meckel's, and rib cartilage, v1b was restricted to the dorsal portion of the fetal rib. This distribution appears to correlate with the portion of the rib which will ultimately ossify, rather than with any of the differentiative states of chondrocytes. Together these results suggest that alternative splicing within the amino terminal domain of the alpha1(XI) chain may contribute to the function of type XI collagen and that expression of the basic v1b peptide may play a role in endochondral ossification.
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PMID:Temporal and spatial expression of alternative splice-forms of the alpha1(XI) collagen gene in fetal rat cartilage. 973 97

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pair-fed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs. that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity.
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PMID:Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex. 1008 58

Mimecan is a proteoglycan expressed by many connective tissues. It was originally isolated in a truncated form as a bone-associated glycoprotein, osteoglycin, and was considered an osteoinductive factor. Recently, we demonstrated that the full-length translation product of the cDNA encoding mimecan is a corneal keratan sulfate proteoglycan present in other tissues without keratan sulfate chains. We also described multiple mimecan mRNA transcripts generated by differential splicing and alternative polyadenylation. In this study, we isolated genomic clones and determined the genomic organization of the bovine mimecan gene. The gene is spread over >33 kilobases of continuous DNA sequence and contains eight exons. The newly discovered first exon, identified by 5'-rapid amplification of cDNA ends, consists of a 5'-untranslated region and is enriched in C+G nucleotides. Two transcription initiation sites starting at the first and at the second exons were determined by primer extension. Molecular characterization shows that alternatively spliced RNA isoforms are generated by the use of two distinct splice acceptor sites in the third exon situated 278 base pairs apart. We determined a partial genomic structure of the human mimecan gene and demonstrated two alternatively spliced RNA transcripts that are generated likewise. Despite the diversity of mimecan transcripts, the primary structure of the core protein is encoded from exons 3 to 8 and remains unchanged, indicating its functional importance. Using ribonuclease protection assay, we analyzed the patterns of spliced RNA expressed in cultured bovine keratocytes. We demonstrated that their expression is differentially modulated in a temporal manner by basic fibroblast growth factor.
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PMID:The bovine mimecan gene. Molecular cloning and characterization of two major RNA transcripts generated by alternative use of two splice acceptor sites in the third exon. 1037 82

ARD-1 is an endoribonuclease identified initially as the product of a human cDNA that complements mutations in rne, a gene that encodes Escherichia coli ribonuclease E. NIPP-1 was identified in bovine nuclear extracts as an inhibitor of protein phosphatase-1. Earlier work has shown that the protein-coding sequence of ARD-1 is identical to the carboxy-terminal third of NIPP-1. However, whether ARD-1 is present in eukaryotes as a distinct entity has been unclear, as neither ARD-1-specific transcripts nor ARD-1 protein were detected in mammalian cells in earlier studies. Here we show that ARD-1 exists in human cells as a discrete protein, and that the ARD-1 and NIPP-1 peptides are isoforms encoded by a single gene and the same alternatively spliced precursor RNA. A retained intron containing multiple translation stop codons that are configured to terminate translation and initiate nonsense-mediated decay, limits the production of cellular ARD-1 protein. Our results establish the process by which functionally disparate ARD-1 and NIPP-1 peptides are generated from the protein-coding sequence of the same gene in human cells.
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PMID:Alternative splicing regulates the production of ARD-1 endoribonuclease and NIPP-1, an inhibitor of protein phosphatase-1, as isoforms encoded by the same gene. 1056 11

Oncostatin M (OSM) is a member of the IL-6 family of polyfunctional cytokines. The characterized murine OSM transcript consists of three exons and encodes a secreted protein. Investigations of mOSM expression using the ribonuclease protection assay demonstrated novel sites of expression in undifferentiated but not differentiated pluripotent cells, and revealed the existence of alternatively spliced mOSM transcripts. cDNAs representing a novel mOSM transcript (mOSM 13) containing exon 1 spliced directly to exon 3 were isolated from bone marrow using Rapid Amplification of cDNA Ends (RACE) PCR and RT-PCR approaches. Expression of the mOSM 13 transcript was regulated in a tissue-specific manner and independently of mOSM transcript production, suggesting that its production is biologically significant. Splicing of exon 1 directly to exon 3 disrupts the OSM open reading frame of mOSM 13. Initiation of translation at sites within exon 3 of mOSM 13 would yield N-terminally truncated OSM proteins that are localized within the cell. The omission of exon 2 by alternate splicing and the production of intracellular proteins with alternate biological activities are conserved among several IL-6 family cytokines and are one manifestation of a more general phenomenon; the production of alternate cytokine transcripts encoding intracellular and extracellular proteins.
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PMID:Regulated expression of alternate transcripts from the mouse oncostatin M gene: implications for interleukin-6 family cytokines. 1067 Dec 98

The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-aminolevulinate synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents approximately 35-45% of total ALAS2 mRNA while the newly identified transcripts together represent approximately 15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.
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PMID:The major splice variant of human 5-aminolevulinate synthase-2 contributes significantly to erythroid heme biosynthesis. 1464 93

The gene FLT1 produces at least two transcripts from a common transcription start site: full-length Flt1 contains 30 exons encoding a membrane-bound VEGF receptor; soluble Flt1 (sFlt1) shares the first 13 exons but utilizes poly(A) signal sequences within intron 13 to create a transcript that lacks downstream exons. To address the mechanisms that regulate human sFlt1, we mapped the 3' end of sFlt1 mRNA and defined the full extent of its 3' untranslated region (UTR). We identified a 3.2 Kb sFlt1 transcript that is cleaved within an alternatively spliced exon downstream of exon 14 and is predicted to encode a C-terminal variant of sFlt1 with an unusual polyserine tail. sFlt1 mRNA cleavage sites within intron 13 were identified in human placenta and in vascular endothelium by ribonuclease protection assay (RPA). A proximal and two distal mRNA cleavage sites were identified by RPA downstream of consensus polyadenylation signals that create variant transcripts with a 3' UTR ranging from 30 bases to approximately 4 Kb. Northern blot analysis and 3' rapid amplification of cDNA ends (RACE) in placenta confirmed the existence of distal intronic sFlt1 cleavage sites that give rise to a sFlt1 transcript of approximately 7 Kb. The identity of the distal signal sequences were then confirmed by mutagenesis of putative signal elements in a polyadenylation reporter assay. We demonstrate the heterogeneity of human sFlt1 that arises from alternate splicing and from alternative polyadenylation directed by strong intronic poly(A) signal sequences leading to C-terminal variants and to an sFlt1 transcript with a large 3' UTR containing several AU rich elements and poly(U) regions that may regulate mRNA stability.
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PMID:Intronic polyadenylation signal sequences and alternate splicing generate human soluble Flt1 variants and regulate the abundance of soluble Flt1 in the placenta. 1761 62


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