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At least 631 records · Page 35Linked to original sources
PubMed1989

Coexistence of a c-myc mRNA initiated in intron 1 with the normal c-myc mRNA and similar regulation of both transcripts in mammalian cells.

We previously described a c-myc mRNA initiated within intron 1 at a major start site designated P3. This mRNA was observed in tumorigenic murine cells with no detectable rearrangement of the c-myc gene. In the present study, we report that P3 mRNA also coexists with the P1/P2 c-myc transcript in non-tumorigenic murine cell lines and that a c-myc mRNA which appears to be an equivalent of the murine P3 transcript is found in various rat and human cells. These data suggest that P3 represents a promoter of the normal, non-rearranged mammalian c-myc gene. Furthermore, both P1/P2 and P3 mRNA levels are similarly regulated by cycloheximide and by dimethylsulfoxide (DMSO) in murine erythroleukemia Friend cells.

Animals↗
PubMed1981

[Ratio of the amounts of the mRNA poly A+/poly A- fractions in the myocardium and the life span of mRNA poly A+].

Two mRNA fractions, poly A+ and poly A- were found in the rat heart muscle. They comprise 30 and 70% of the entire mRNA, respectively. The ratio of the mRNA poly A+ to poly A- in the myocardium does not change in physiological aging. According to the kinetics of labelled precursor incorporation into mRNA, the life span of the main mass of mRNA poly A+ in the myocardium is about 4 hours.

Aging↗
PubMed1994

Analysis of the role of the Shine-Dalgarno sequence and mRNA secondary structure on the efficiency of translational initiation in the Euglena gracilis chloroplast atpH mRNA.

Chloroplast mRNAs in Euglena gracilis fall into two classes. One class has a Shine-Dalgarno sequence 5' to the AUG start codon while the other group of mRNAs does not have any conserved sequence elements near the start codon. The chloroplast mRNA encoding the atpH gene has been selected as an example of a message which has a Shine-Dalgarno sequence (GGAGUU) located in the initiation region. Mutations in the Shine-Dalgarno sequence result in 2-5-fold reductions in the efficiency of the message in initiation complex formation depending on the precise mutation. Secondary structure mapping of the initiation region of the atpH mRNA suggests that a number of mRNA conformations are present in an equilibrium mixture. Mutations in the Shine-Dalgarno sequence had little effect on the overall structure of the mRNA. Directed mutations which place the Shine-Dalgarno sequence into a region of higher secondary structure drastically reduce the activity of the message. The monocistronic form of the atpH mRNA has a 5'-untranslated leader region slightly over 70 nucleotides in length. Deletions in the leader suggest that a minimum length of 10 bases 5' to the start codon is important for activity in initiation. The presence of the full leader increased efficiency in initiation about 2-fold compared to messages with leaders 25-40 residues in length.

Animals↗
PubMed1994

A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

Amino Acid Sequence↗
PubMed1994

Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex.

N6-Methyladenosine is found at internal positions of mRNA in higher eukaryotes. This post-transcriptional modification occurs at a frequency of one to three methylation/average mRNA molecule in mammalian cell lines and is sequence-specific. A highly conserved consensus recognition site for the methyltransferase has been determined from both viral and cellular messages, consisting of the sequence Pu(G/A)AC(U/A) (with A being methylated). Despite the ubiquity and the specificity of this modification, little is known about the mechanism of formation of N6-methyladenosine. Utilizing an in vitro methylation system from HeLa cell nuclear extracts, and a substrate RNA derived from the mRNA coding for bovine prolactin, the mRNA N6-adenosine methyltransferase has been characterized and partially purified. Unique among other characterized nucleic acid methyltransferases, the enzyme is composed of three components which are separable under non-denaturing conditions. The molecular masses of the components are 30, 200, and 875 kDa as determined by gel filtration and glycerol gradient sedimentation. The 200-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit. The 875-kDa component has affinity for single-stranded DNA-agarose, suggesting that it may contain the mRNA-binding site. N6-Adenosine methyltransferase is not sensitive to treatment with micrococcal nuclease, nor to immunodepletion using an anti-trimethylguanosine antibody, suggesting that it does not contain an essential RNA component.

Animals↗
PubMed2025

Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling.

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon.

RNA Stability↗
PubMed1998

Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme.

Current models of mRNA decay in yeast posit that 3' deadenylation precedes enzymatic removal of the 5' cap, which then exposes the naked end to 5' exonuclease action. Here, we analyzed gene expression in Saccharomyces cerevisiae cells bearing conditional mutations of Ceg1 (capping enzyme), a 52 kDa protein that transfers GMP from GTP to the 5' end of mRNA to form the GpppN cap structure. Shift of ceg1 mutants to restrictive temperature elicited a rapid decline in the rate of protein synthesis, which correlated with a sharp reduction in the steady-state levels of multiple individual mRNAs. ceg1 mutations prevented the accumulation of SSA1 and SSA4 mRNAs that were newly synthesized at the restrictive temperature. Uncapped poly(A)+ SSA4 mRNA accumulated in cells lacking the 5' exoribonuclease Xrn1. These findings provide genetic evidence for the long-held idea that the cap guanylate is critical for mRNA stability. The deadenylation-decapping-degradation pathway appears to be short-circuited when Ceg1 is inactivated.

Exoribonucleases↗
PubMed1975

Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.

The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA (guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl- and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an S20,w of 6.0 and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations. Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system.

Guanine Nucleotides↗
PubMed1996

Amplification of specific mRNA from a single human renal glomerulus, with an approach to the separation of epithelial cell mRNA.

A method has been developed by which single human glomeruli may be plucked from fresh renal biopsies under direct vision, followed by separation of mRNA using oligo-dT-linked paramagnetic beads. The mRNA was amplified by reverse transcription and polymerase chain reaction (RT-PCR). Primers for a variety of human and rat proteins have been developed. The quantity of the amplified cDNA was measured by an enzyme-linked immuno-sorbent assay (ELISA), where biotinylated forward strands of DNA were captured, probed with a fluorescein-conjugated DNA oligomer, and then assayed with an enzyme-linked anti-fluorescein antibody. The cDNA-linked beads are reported to be stable and can be reused with different primer sets, thus forming a "bank' of samples from cases with defined glomerular disorders, which can be used to address new questions as they arise. Using rat glomeruli, a method has been devised which permits at least partial separation of epithelial cell mRNA from mesangial and endothelial cell mRNA.

Actins↗
PubMed1996

Developmental expression and CORT-regulation of TGF-beta and EGF receptor mRNA during mouse palatal morphogenesis: correlation between CORT-induced cleft palate and TGF-beta 2 mRNA expression.

Glucocorticoids (CORT) have been shown to induce cleft palate in mice. Although the pathogenetic pathway of CORT-induced cleft palate has been investigated for several decades, the molecular details remain to be elucidated. Since growth factors have been shown to regulate palate morphogenesis, and the expression of several growth factors or their receptors, e.g. TGF-beta, EGF receptor (EGF-R), are known to be modulated by CORT, we postulate that CORT modulation of growth factor (or receptor) gene expression is a key mechanism involved in CORT-induced cleft palate. To test this hypothesis, we analyzed the steady-state levels (Northern and RNase protection) and developmental expression (in situ hybridization) of four CORT-responsive genes--TGF-Beta 1, TGF- beta 2, TGF-beta 3, and EGF receptor (EGF-R)--in developing mouse palates in the presence or absence of exogenous CORT. Pregnant B10.A dams were injected on day 12 of gestation with CORT or sham-injected and embryonic palates were collected at 1, 2, and 3 days postinjection (E13-E15). During mouse palate development, significant increases in TGF-beta 1 and TGF-beta 3 mRNA levels, as well as significant decrease in TGF-beta 2 mRNA levels, are detected; no significant difference in EGF-R transcript level is observed with progressive development. In CORT-exposed palates, we demonstrate no significant differences in the direction or magnitude of change with time in TGF-beta 1, TGF-beta 3, and EGF-R mRNA levels compared to controls. However, CORT delays by 1 day the down-regulation of palatal TGF-beta 2 transcript normally seen on day 14 of gestation. TGF-beta 2 is known to inhibit cell proliferation. The level of TGF-beta 2 mRNA, the only isoform primarily expressed in the palatal mesenchyme, significantly decreases with progressive palatal development; this down-regulation of TGF-beta 2 expression is associated with increased mesenchymal cell proliferation and palatal shelf growth. CORT, at a critical stage of palatogenesis, induces a delay in the normal down-regulation of TGF-beta 2 gene expression. Given that CORT is known to inhibit mesenchymal cell proliferation and palatal shelf growth, we conclude that the CORT-induced delay in the normal down-regulation of TGF-beta 2 gene expression is probably key event in the pathogenesis of CORT-induced cleft palate.

Animals↗
PubMed2000

Temporal analysis of growth factor mRNA expression in myelinating rat brain aggregate cultures: increments in CNTF, FGF-2, IGF-I, and PDGF-AA mRNA are induced by antibody-mediated demyelination.

Myelinogenesis in rat brain aggregate cultures is associated with a pattern of growth factor mRNA expression comparable to that of the developing brain. The rate of increase in platelet-derived growth factor-AA (PDGF-AA) expression was greatest just before the detection of myelin basic protein (MBP) mRNA in the cultures and remained high thereafter, consistent with in vivo observations. Levels of fibroblast growth factor-2 (FGF-2) and of ciliary neurotrophic factor (CNTF) mRNA increased continuously over the period of MBP accumulation. High rates of transforming growth factor beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and neurotrophin-3 (NT-3) expression at early time points during the culture gradually decreased over time, indicative of a key regulatory role during oligodendrocyte development. The addition of demyelinative anti-myelin oligodendrocyte glycoprotein (anti-MOG) antibody resulted in a significant increase in MBP peptide fragments with a C-terminus at phenylalanine 89 indicating proteolytic breakdown of MBP after myelin phagocytosis. Immediately after antibody treatment the expression of CNTF mRNA was significantly increased, compared with controls, while that of FGF-2 and IGF-I, and of PDGF-AA peaked during the early and later stages of recovery respectively. Thus, specific growth factors combine to regulate myelination and remyelination in the aggregates; these data have implications for demyelinating disease in which protective growth factor secretion may be central to regeneration.

Animals↗
PubMed2003

Expressions of human telomerase mRNA component (hTERC) and telomerase reverse transcriptase (hTERT) mRNA in effusion cytology.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA onto chromosomal ends. Its activity has been reported to be a novel diagnostic marker of malignant potential. However, no study has investigated the diagnostic importance of the expressions of human telomerase mRNA component (hTERC) and telomerase reverse transcriptase (hTERT) mRNA in effusion cytology. We investigated the expressions of hTERC and hTERT using in situ hybridization in 20 malignant cytologic specimens from 20 patients with adenocarcinomas disseminating into the pleura or peritoneum, and 16 benign cytologic specimens in pleural or peritoneal fluid (including reactive mesothelial cells) from 16 patients with non-neoplastic diseases. Expressions of hTERC and hTERT mRNA were detected in 95% and 90% of the 20 malignant cytology specimens, respectively, and were apparent within the cytoplasm of malignant cells. However, no expression was detected in reactive mesothelial cells in the 16 benign cytologic specimens. The expressions of hTERC and hTERT mRNA would appear to be a useful addition to cytology examination for diagnosis based on effusion cytology.

Adenocarcinoma↗
PubMed1990

A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism.

We have replaced the 14 nucleotide long intervening sequence of the Saccharomyces cerevisiae SUP6 (ochre) tRNA(Tyr) gene by the 52 nucleotide long second intron of the S. cerevisiae MATa1 gene. Yeast cells containing this modified pre-tRNA showed the typical suppressor phenotype indicating that the MATa1 pre-mRNA intron was exactly excised in vivo from the primary tRNA transcript and a mature and functional tRNA was formed. Several lines of evidence show that the splicing reaction proceeded via the pre-mRNA splicing mechanism: the reaction yielded a lariat shaped excised intron with a lariat shaped intron-exon 2 molecule as intermediate; point mutations in the conserved UAC-UAAC box of the intron impaired splicing of the precursor RNA; in a temperature sensitive rna2 strain splicing of this tRNA precursor was inhibited at the restrictive temperature. Our results imply that in yeast the excision of a pre-mRNA intron is not dependent on the transcription apparatus by which it was generated and that transcription and splicing are uncoupled processes in vivo, too. Furthermore these data demonstrate that recognition of an RNA as a substrate for a pre-mRNA splicing reaction is, at least qualitatively, only intron dependent.

Base Sequence↗
PubMed1991

The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon.

Messenger RNA molecules 30-35 bases long, with sequences related to the 5'-region of cro-mRNA from lambda-phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio-UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross-linked ribosomal proteins were identified immunologically, and cross-linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio-U residue involved in the cross-link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross-linked complex. The principal results were that, at thio-U positions upstream of the AUG codon, specific cross-linking occurred to protein S7 and to the 3'-terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross-linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA-dependent cross-link was found to position approximately 1050 of the 16S RNA, but--in contrast to similar experiments with 70S ribosomes--no cross-linking was found to the 1390-1400 region.

Base Sequence↗
PubMed1998

IL-10 suppresses LPS-induced KC mRNA expression via a translation-dependent decrease in mRNA stability.

This study examines the mechanism of interleukin-10 (IL-10)-mediated suppression of KC chemokine gene expression in mouse macrophages. Suppression of KC mRNA levels by IL-10 occurred late in the time course of response to lipopolysaccharide (LPS). Equivalent IL-10-mediated suppression was observed when the agent was added 1 h before, simultaneous with, or 1 h after LPS. IL-10 did not inhibit KC gene transcription but rather produced a decrease in the stability of KC mRNA. The suppressive action of IL-10 was prevented in macrophages that were also treated with inhibitors of protein synthesis even when added 2 h after LPS and IL-10. These results suggest that IL-10 acts to destabilize LPS-induced KC mRNA through a process that depends on coincident KC mRNA translation.

Animals↗
PubMed1993

Ovariectomy decreases the mRNA levels of transforming growth factor-beta 1 and increases the mRNA levels of osteocalcin in rat bone in vivo.

Estrogen depletion causes postmenopausal osteoporosis. Here we report that steady state mRNA levels of transforming growth factor-beta 1 (TGF-beta 1) and osteocalcin in bone persistently decreased and increased, respectively, in vivo in estrogen-depleted rats after ovariectomy (OVX). 21 female Wistar rats (7-month-old) were randomized and underwent OVX or sham-operation, total RNA was extracted from tibiae and assessed by Northern blot analysis. OVX induced 70-80% decrease in TGF-beta 1 mRNA levels and 2- to 3-fold increase in mRNA levels of osteocalcin compared with controls three weeks after surgery. These changes persisted up to twelve weeks post-operation. OVX caused 15% reduction in femoral bone mineral density and 2-fold elevation in serum osteocalcin levels as early as two weeks post-operation. Moreover, estrogen depletion resulted in marked decrease and increase, respectively, in steady state mRNA levels of TGF-beta 1 and osteocalcin in vitro in osteoblastic rat osteosarcoma cells, ROS 17/2.8. Our results provide the first in vivo evidence that expression of TGF-beta and osteocalcin in bone is reciprocally regulated at the transcriptional level in estrogen deficient OVX rats and suggests that TGF-beta 1 may play a role in estrogen-dependent maintenance of normal bone density.

Amino Acid Isomerases↗
PubMed1995

Pyrrolidine dithiocarbamate prevents IL-1-induced nitric oxide synthase mRNA, but not superoxide dismutase mRNA, in insulin producing cells.

We presently investigated the effects of pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor kappa B (NF-kappa B), on the induction of nitric oxide synthase (iNOS) and manganese superoxide dismutase (MnSOD) mRNAs by IL-1 beta in insulin-producing RIN cells. PDTC decreased by 90% both IL-1 beta-induced increase in medium nitrite accumulation (an indicator of NO production) and induction of iNOS mRNA expression. However, PDTC did not prevent induction of MnSOD mRNA by IL-1 beta. PDTC induced an early (45 min) expression of c-fos mRNA and potentiated IL-1 beta-induced c-fos expression. Our data suggest that NF-kappa B activation is necessary for iNOS, but not for MnSOD, mRNA expression in insulin producing cells.

Amino Acid Oxidoreductases↗
PubMed1999

PPARalpha activation by Wy 14643 induces transactivation of the rat UCP-1 promoter without increasing UCP-1 mRNA levels and attenuates PPARgamma-mediated increases in UCP-1 mRNA levels induced by rosiglitazone in fetal rat brown adipocytes.

Rodent brown adipocytes express peroxisome proliferator activated receptor-alpha (PPARalpha) and PPARgamma and while the rodent uncoupling protein-1 (UCP-1) gene contains a putative peroxisome proliferator response element (PPRE), only PPARgamma activation by thiazolidinediones increase UCP-1 mRNA levels. We have investigated this phenomenon in foetal rat brown adipocytes (FBA) and show that although transactivation occurs in FBA containing a plasmid encoding 4.5kb of the 5'-flanking region of the rat UCP-1 promoter ((-4551)-UCP-1-CAT) treated with either the selective PPARgamma agonist rosiglitazone (1.0 microM) or the selective PPARalpha agonist Wy 14643 (10.0 microM), only rosiglitazone induced transcription of UCP-1 mRNA. Furthermore, Wy 14643 (10 and 100.0 microM) abolished rosiglitazone-induced UCP-1 mRNA induction in spite of a transactivation event occurring with the combination treatment. Thus in FBA PPARalpha-activation with Wy 14643 elicits a "blind" transactivation of the UCP-1 promoter which can prevent PPARgamma-mediated UCP-1 mRNA transcription either by competition for the PPRE or by an unidentified post-transcriptional event.

Adipose Tissue, Brown↗