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At least 991 records · Page 55Linked to original sources
PubMed1997

Ethanol exposure affects glial fibrillary acidic protein gene expression and transcription during rat brain development.

Exposure to ethanol during fetal development reduces the astroglial-specific marker glial fibrillary acidic protein (GFAP) and its mRNA levels in brains of fetal rats and in radial glia in primary culture, affecting the proliferation and differentiation of astrocytes. The objectives of this study were to evaluate the possible effect of ethanol on GFAP mRNA levels in astrocytes and to investigate the molecular mechanism(s) involved in ethanol-induced changes in GFAP expression by analyzing the GFAP transcription rate, GFAP mRNA stability, and GFAP DNA methylation. We show here that prenatal exposure to ethanol reduces significantly GFAP immunoreactivity and its mRNA levels in both astrocytes in primary culture and brains of pups from alcohol-fed mothers. Runoff experiments from nuclei of astrocytes indicate that ethanol exposure decreases GFAP transcription rate significantly and reduces GFAP mRNA stability slightly. DNA methylation analysis indicates that prenatal ethanol exposure induces a hypermethylated state of the GFAP DNA in fetal brains. Methylation-mediated repression of GFAP transcription could be a mechanism involved in ethanol-induced reduction of GFAP expression. Ethanol-induced alterations in GFAP expression and astroglial development may underlie the CNS dysfunctions observed after prenatal alcohol exposure.

Aging↗
PubMed1988

Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor beta, and cAMP in human cell lines.

The regulation of fibronectin (FN) biosynthesis by dexamethasone (a synthetic glucocorticoid), forskolin (an activator of adenylate cyclase), and transforming growth factor beta (TGF-beta) was examined in six human cell lines. Dexamethasone treatment produced the largest increase in FN biosynthesis in the fibrosarcoma cell line, HT-1080 (approximately 45-fold). This seems to result from a dexamethasone-mediated increase in FN mRNA stability which increases the message half-life from approximately 11 to 26 h. The relative instability of FN mRNA in the fibrosarcoma (t1/2 11 h) compared to normal fibroblasts (70 h) appears to result from the particular transformed phenotype of the HT-1080 cells. Forskolin and TGF-beta increase the rate of FN gene transcription in most of the cell lines. These effects (four- to six-fold) occur rapidly and do not require protein synthesis in the responsive cell lines which include normal fibroblasts. However, in the fibrosarcoma (HT-1080), a surprisingly large induction (20-30-fold) is observed and this induction is different from that in the normal fibroblasts and the other cell lines in that both protein synthesis and a lag period are required. Synergism is seen with dexamethasone and either forskolin or TGF-beta in HT-1080 cells increasing the rate of FN biosynthesis approximately 200-fold to a level similar to normal fibroblasts. This seems to result from a combination of FN mRNA stabilization (dexamethasone) and increased transcription (forskolin and TGF-beta).

1-Methyl-3-isobutylxanthine↗
PubMed1998

Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin 1alpha, tumor necrosis factor-alpha, glucocorticoids, and 17beta-estradiol.

OBJECTIVE: To determine the effects of interleukin 1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), dexamethasone, and 17beta-estradiol on the expression of cyclooxygenase-1 (COX-1) and COX-2 in bovine chondrocytes. METHODS: Northern blot analysis was used to quantify COX-1 and COX-2 mRNA expression in primary cultures of bovine chondrocytes and prostaglandin production to evaluate COX activity. RESULTS: IL-1alpha and TNF-alpha increased the expression of COX-2. This effect was independent of de novo protein synthesis and dependent on increased mRNA stability in the case of IL-1alpha. Dexamethasone inhibited the effects of both cytokines. 17beta-estradiol inhibited COX-2 mRNA expression in basal conditions, but had no effect on COX-2 expression induced by cytokines. The specific COX-2 inhibitor compound NS 398 prevented the increase in prostaglandin E2 (PGE2) production induced by the cytokines. COX-1 levels remained stable with all treatments. CONCLUSION: Increase in mRNA stability is a mechanism implicated in the induction of COX-2 by some cytokines. The effects of IL-1alpha and TNF-alpha on PGE2 production are mainly due to an increase in COX-2 activity as shown by the effect of compound NS 398. 17beta-estradiol inhibits COX-2 mRNA expression in basal conditions, suggesting that estrogens could be implicated in the control of cartilage metabolism.

Animals↗
PubMed1999

Chronic ethanol consumption leads to destabilization of rat liver beta-galactoside alpha2,6-sialyltransferase mRNA.

Chronic ethanol consumption in rats is accompanied by decreased levels of Gal beta1,4GlcNAc alpha2,6-sialyltransferase (2,6-ST) activity in the liver. Our previous studies have shown that there is a concomitant decrease in the levels of 2,6-ST mRNA. In this study, the alteration in the regulation of 2,6-ST expression by chronic ethanol consumption was assessed by Northern hybridization, nuclear run-on experiments, and 2,6-ST mRNA stability studies. 2,6-ST downregulation was found at 4 weeks of feeding an ethanol diet (36% of calories from ethanol) and remained up to 8 weeks. The decrease in 2,6-ST mRNA levels was found to be dose-dependent, with lower dose of ethanol (12% and 24% of total dietary calories from ethanol) being ineffective and the effects being manifested only when 36% of the dietary calories were from ethanol. The effects of chronic ethanol feeding could be completely reversed within 1 week after ethanol consumption was stopped, when 2,6-ST mRNA levels were restored to normal. The downregulation was not sensitive to actinomycin D, indicating that the regulation was not affected at the transcriptional level but at the posttranscriptional level. This was confirmed by nuclear run-on experiments showing that the rate of 2,6-ST mRNA transcription was unaffected by ethanol. Finally, mRNA stability experiments showed that the half-life of 2,6-ST mRNA was reduced 50% in ethanol-fed rat livers compared with control rat livers. Taken together, the results show that 2,6-ST mRNA is regulated at the posttranscriptional level and chronic ethanol intake downregulates 2,6-ST expression by destabilizing its mRNA.

Animals↗
PubMed1987

The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site.

Various synthetic DNA sequences were inserted downstream of the fourth codon of the Escherichia coli lacZ gene on plasmids containing a hybrid lacZ-galK operon. Several different sequences, one as short as 10 bp, reduced the functional stability of the lacZ message three- to fourfold, whereas others had little or no effect. Introduction of synthetic sequences into a plasmid containing the intact lac operon resulted in similar reductions of mRNA stability. The sequence alterations also reduced the translational efficiency and transcription through lacZ as monitored by measurements of galactokinase synthesis from the downstream galK gene. There was no correlation between the average translational frequency and the stability of the lacZ message indicating that some of the inserted sequences reduced mRNA stability directly and not as a consequence of their effect on translation. The reduction of transcription through the lacZ gene correlated with the reduction of translation in agreement with current models of transcriptional polarity.

Amino Acid Sequence↗
PubMed1996

Differential stability of zein mRNA in developing corn kernel.

The lifetime of the zein mRNA in a developing corn (Zea mays L.) kernel under genome transcription blockade with actinomycin D (in vivo) and in a cell-free system (in vitro) was studied. After a 10 h blockade of gene transcription with actinomycin D, only 55% of 19 kDa zein mRNA and 40% of 22 kDa mRNA were detected in a developing kernel of normal corn. In that of the opaque-2 mutant 80% of 19 kDa zein mRNA remained. To examine the relative stability of poly(A)-containing mRNA, cell-free systems from rabbit reticulocyte lysate and wheat-germ extract were used. In both cases only 40% of 19 kDa zein mRNA and 60% of 22 kDa zein mRNA decayed during a 30 min incubation. Differential mRNA degradation of poly(A)-containing zein mRNA was observed on affinity chromatography; poly(A)-containing 19 kDa zein mRNA from normal corn partially decayed by elution from poly(U)-Sepharose whereas that from opaque-2 remained stable. These data suggest that differential mRNA stability is an important factor in the regulation of the zein gene expression in a developing corn kernel.

Animals↗
PubMed1997

Transcriptional and post-transcriptional regulation of LDL receptor gene expression in PMA-treated THP-1 cells by LDL-containing immune complexes.

We have previously shown that uptake of low density lipoprotein-containing immune complexes (LDL-IC) by human monocyte-derived macrophages led to the transformation of these cells into foam cells and induced a paradoxical increase in receptor-mediated binding of 125I-labeled LDL due to an increase in the number of LDL receptors (LDL-R). The same metabolic changes are also observed in PMA-treated THP-1 cells after incubation for 2 h with 150 microg/ml of immune complexes containing either native, oxidized (ox), or malondialdehyde (mda) LDL. After stimulation, PMA-treated THP-1 cells showed not only a 40-fold increase in 125I-labeled LDL binding but also a 40-fold increase in the immunoreactive LDL-R protein, confirming that the increase in LDL binding is due to an increase LDL-R number. In this study we have investigated, in PMA-treated THP-1 cells, the regulatory mechanism(s) responsible for the increased receptor-mediated binding of LDL induced by LDL-IC. By Northern blot and nuclear run-on analysis we have shown transcriptional activation of the LDL-R gene with a 7-fold increase in the LDL-R mRNA level in LDL-IC stimulated cells. Due to the marked difference between the increase in LDL-R mRNA and LDL-R protein, we estimated LDL-R mRNA stability using a inhibitor chase method and have shown that LDL-IC did not alter the LDL-R mRNA stability in THP-1 cells. We have also demonstrated, using cycloheximide as a inhibitor of protein synthesis, that the marked increase in LDL-R protein observed in LDL-IC-stimulated THP-1 cells resulted from de novo synthesis of LDL-R protein. To determine whether the increase in transcriptional activity of the LDL-R gene was secondary to changes in the cholesterol regulatory pool we performed experiments in which the cell cholesterol content was modified by the addition of either 25-hydroxycholesterol and mevalonate or inhibitors of ACAT activity (SA-58035 and progesterone). These experiments showed that the enhanced LDL-R expression was not affected by the addition of any of the above compounds. In conclusion, LDL-IC induced both transcriptional and post-transcriptional activation of the LDL-R gene in PMA-treated THP-1 cells and this induction was independent of the free cholesterol content of these cells.

Antigen-Antibody Complex↗
PubMed1995

Post-transcriptional regulation of the stanniocalcin gene by calcium.

Stanniocalcin (STC) is a Ca(2+)-regulating hormone produced by the corpuscles of Stannius in bony fish. Calcium has been shown to stimulate STC synthesis at multiple levels including the level of gene expression. The purpose of this study was to determine the effects of Ca2+ on STC mRNA stability. The half-life of STC mRNA was measured in primary cultured trout corpuscles of Stannius cells maintained in either normal (1.2 mM) or high (1.9 mM) levels of extracellular calcium and treated with the transcriptional inhibitor alpha-amanitin. In cells maintained in 1.2 mM Ca2+, STC mRNA levels decreased progressively over time with an estimated half-life of approximately 71 h. However, message levels remained unchanged for up to 4 days in cells maintained in 1.9 mM Ca2+, indicating that the transcript had been stabilized in response to Ca2+ stimulation. Blocking transcription prior to exposing cells to high Ca2+ did not alter the stabilizing effects of the cation, indicating that synthesis and processing of the mRNA transcript were not involved in message stabilization. Inhibiting protein synthesis with cycloheximide also had no influence on the stabilizing effects of high calcium. The experiments involving cycloheximide further suggested that the mechanism of mRNA stabilization involved protein-nucleic acid interactions in the cytoplasm, whereby the polysomal complex protected the mRNA from degradation. These data demonstrate that the stimulatory effect of Ca2+ on STC gene expression is due, in part, to mRNA stabilization.

Amanitins↗
PubMed2006

Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on log-phase mRNA half-lives.

Bacterial pathogens regulate virulence factor expression at both the level of transcription initiation and mRNA processing/turnover. Within Staphylococcus aureus, virulence factor transcript synthesis is regulated by a number of two-component regulatory systems, the DNA binding protein SarA, and the SarA family of homologues. However, little is known about the factors that modulate mRNA stability or influence transcript degradation within the organism. As our entree to characterizing these processes, S. aureus GeneChips were used to simultaneously determine the mRNA half-lives of all transcripts produced during log-phase growth. It was found that the majority of log-phase transcripts (90%) have a short half-life (<5 min), whereas others are more stable, suggesting that cis- and/or trans-acting factors influence S. aureus mRNA stability. In support of this, it was found that two virulence factor transcripts, cna and spa, were stabilized in a sarA-dependent manner. These results were validated by complementation and real-time PCR and suggest that SarA may regulate target gene expression in a previously unrecognized manner by posttranscriptionally modulating mRNA turnover. Additionally, it was found that S. aureus produces a set of stable RNA molecules with no predicted open reading frame. Based on the importance of the S. aureus agr RNA molecule, RNAIII, and small stable RNA molecules within other pathogens, it is possible that these RNA molecules influence biological processes within the organism.

Bacterial Proteins↗
PubMed1990

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12.

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.

Escherichia coli↗
PubMed2002

Mechanisms of the regulation of EGF receptor gene expression by calcitriol and parathyroid hormone in UMR 106-01 cells.

BACKGROUND: We have previously demonstrated that parathyroid hormone (PTH) and calcitriol increase the expression of epidermal growth factor receptors (EGFR) in UMR 106-01 osteoblast-like cells. The effect of PTH is mediated by cAMP and it involves an increase in the level of EGFR mRNA. The present studies were designed to investigate the mechanisms involved in the regulation of EGFR expression by PTH and calcitriol. METHODS: To examine the mechanism of the effect of calcitriol on EGFR expression, confluent cultures of UMR 106-01 cells were exposed to calcitriol and levels of EGFR mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). In order to study the effect of calcitriol on EGFR gene transcription, a candidate vitamin D-responsive element (VDRE) was identified in the EGFR gene promoter and complimentary 30-mer oligonucleotides spanning this region were tested for binding to recombinant VDR using EMSA. Transcriptional activity in response to calcitriol and PTH was tested in UMR 106-01 cells stably transfected with a luciferase reporter construct containing the full length EGFR gene promoter. The effect of calcitriol on EGFR mRNA stability was examined in transcriptionally arrested cells. RESULTS: Treatment with calcitriol resulted in a time and dose dependent increase in EGFR mRNA levels in confluent cultures of UMR 106-01 osteoblast-like cells. Using EMSA, we demonstrated that the putative human EGFR VDRE binds to recombinant VDR in a retinoid X receptor (RXR)-dependent manner; however, calcitriol failed to increase transcriptional activity from a luciferase reporter construct containing the full-length EGFR gene promoter in stably transfected UMR 106-01 cells. Therefore, EGFR mRNA degradation was examined in transcriptionally arrested cells and calcitriol was found to prolong the half life of EGFR mRNA. Treatment of the cultures with PTH resulted in a ninefold increase in luciferase activity after four hours of exposure, a finding that was reproduced by treatment with forskolin. CONCLUSIONS: These studies demonstrate that the calciotropic hormones PTH and calcitriol increase EGF receptor expression by different mechanisms. The former increases EGFR gene transcription whereas the latter increases EGFR mRNA stability.

Calcitriol↗
PubMed1992

Matrix metalloproteinases in periodontal tissue remodelling.

Inflammation of the periodontium leads to connective tissue degradation and eventual tooth loss. The regulation of matrix metalloproteinases (MMPs) has been studied to determine their role in these processes and also during tissue remodelling. Analysis of gingival crevicular fluid has revealed the presence of collagenase and gelatinase that, in the acute stages of periodontal disease, are derived predominantly from polymorphonuclear leukocytes. These MMPs appear to be intimately associated with tissue destruction since the levels of the active forms of these enzymes obtained from either crevicular fluid or mouthrinse samples correlate with tissue destruction and, therefore, provide a sensitive means of demonstrating disease activity. Transforming growth factor-beta, an important regulator of connective tissue remodelling, has been implicated in the rapid remodelling of periodontal tissues. TGF-beta promotes tissue matrix formation by stimulating both the synthesis of matrix proteins (collagen, fibronectin and SPARC) and proteinase inhibitors (TIMP, PAI-1) and by decreasing the synthesis of MMPs, but not the 72 kDa-gelatinase. Nuclear run-on analyses have shown that TGF-beta reduces collagenase and stromelysin synthesis by suppressing gene transcription without altering mRNA stabilities. In contrast, the transcription of the gelatinase and TIMP genes was increased by TGF-beta, which also increased gelatinase mRNA stability. Remodelling of alveolar bone involves interaction between osteoblasts and osteoclasts. Osteoblasts, under the influence of osteotropic hormones (vit D3, PTH and retinoic acid), produce MMPs which appear to function in the removal of soft tissue that precludes access of osteoclasts to the mineralized tissue surface. Rat osteoblastic cells produce MMPs with activity on native collagen, native collagen 3/4-fragments and gelatin and, in addition, two forms of TIMP activity. The 3/4-collagen endopeptidase, purified to apparent homogeneity, also has significant collagenase and gelatinase activities and an amino terminal sequence almost identical to human 72 kDa-gelatinase. The production of this enzyme was stimulated by TGF-beta, which suppresses bone resorption, and by osteotropic hormones which stimulate bone resorption, supporting a bifunctional role for the gelatinase in connective tissue remodelling. Although there is strong evidence for the involvement of MMPs in the resorption of bone and in the inflammation-mediated destruction of periodontal tissues, the role of MMPs in the remodelling of mature soft connective tissues remains equivocal.

Alveolar Bone Loss↗
PubMed1997

Induction of interleukin-2 receptor alpha gene by delta9-tetrahydrocannabinol is mediated by nuclear factor kappaB and CB1 cannabinoid receptor.

Previously, we reported that the cannabinoid delta9-tetrahydrocannabinol (THC) increased the expression of interleukin-2 (IL-2) receptor (R) alpha and beta proteins and mRNAs in NKB61A2 cells, but decreased the level of the gamma-chain message. The drug increased beta-chain message stability rather than increased transcription. In the present study, we examined the mechanism responsible for the drug-induced increase in alpha-chain message in NKB61A2 cells. Nuclear run-on and mRNA stability studies showed THC increased the level of alpha gene transcription but had no effect on mRNA stability. Because expression of this gene is regulated by nuclear factor (NF)-kappaB, we next tested the drug effect on the nuclear level of this protein using the electromobility shift assay. These studies showed a drug-induced increase in NF-kappaB activity. To link the increased nuclear factor activity with the THC-induced increase in IL-2R alpha message, antisense oligodeoxynucleotides were used to inhibit expression of the RelA component of NF-kappaB. These results showed anti-RelA antisense eliminated the cannabinoid-induced upregulation of both alpha mRNA and RelA protein. Furthermore, inhibition of the cannabinoid receptor type 1 with antisense oligomers also eliminated the drug effect on the alpha message. These results suggest that THC treatment of NKB61A2 cells increases IL-2R alpha gene transcription by increasing the nuclear level of NF-kappaB through a mechanism involving cannabinoid receptor type 1 expression.

Animals↗
PubMed2006

The 3'-untranslated region of the Ste20-like kinase SLK regulates SLK expression.

Ste20-like kinase, SLK, a germinal center kinase found in kidney epithelial cells, signals to promote apoptosis. Expression of SLK mRNA and protein and kinase activity are increased during kidney development and recovery from ischemic acute renal failure. The 3'-untranslated region (3'-UTR) of SLK mRNA contains multiple adenine and uridine-rich elements, suggesting that 3'-UTR may regulate mRNA stability. This was confirmed in COS cell transient transfection studies, which showed that expression of the SLK open-reading frame plus 3'-UTR mRNA was reduced by 35% relative to the open-reading frame alone. To further characterize the SLK-3'-UTR, this nucleotide sequence was subcloned downstream of enhanced green fluorescent protein (EGFP) cDNA. In COS, 293T, and glomerular epithelial cells, expression of EGFP mRNA and protein was markedly reduced in the presence of the SLK-3'-UTR. After transfection and subsequent addition of actinomycin D, EGFP mRNA remained stable in cells for at least 6 h, whereas EGFP-SLK-3'-UTR mRNA decayed with a half-life of approximately 4 h. A region containing five AUUUA motifs within the SLK-3'-UTR destabilized EGFP mRNA. Deletion of this region from the SLK-3'-UTR, in part, restored mRNA stability. By UV cross-linking and SDS-PAGE, the SLK-3'-UTR bound to protein(s) of approximately 30 kDa in extracts of COS cells, glomerular epithelial cells, and kidney. Cotransfection of HuR (a RNA binding protein of approximately 30 kDa) increased the steady-state mRNA level of EGFP-SLK-3'-UTR but not EGFP. Thus the SLK-3'-UTR may interact with kidney RNA-binding proteins to regulate expression of SLK mRNA during kidney development and after ischemic injury.

3' Untranslated Regions↗
PubMed1997

Rare codons are not sufficient to destabilize a reporter gene transcript in tobacco.

In plants, as in other eukaryotes, most synonymous codons of the genetic-code are not used with equal frequency, but instead some codons are preferred, whereas others are rare. Circumstantial evidence led to the suggestion that rare codons have a negative influence on mRNA stability. To address this question experimentally, rare codons encoded by a Bacillus thuringiensis (B.t.) toxin gene (cryIA(c)) or a synthetic sequence were introduced into a phytohemagglutinin (PHA) reporter gene. In neither case was the mRNA stability appreciably diminished in stably transformed tobacco cell cultures nor was the accumulation of mRNA in transgenic plants affected. Thus rare codons do not appear to be sufficient to cause rapid degradation of the PHA mRNA and potentially other mRNAs in plants.

Cell Line↗
PubMed2006

Shared stabilization functions of pyrimidine-rich determinants in the erythroid 15-lipoxygenase and alpha-globin mRNAs.

The poly(C)-binding proteins, alphaCPs, comprise a set of highly conserved KH-domain factors that participate in mRNA stabilization and translational controls in developmental and viral systems. Two prominent models of alphaCP function link these controls to late stages of erythroid differentiation: translational silencing of 15-lipoxygenase (Lox) mRNA and stabilization of alpha-globin mRNA. These two controls are mediated via association of alphaCPs with structurally related C-rich 3'-untranslated region elements: the differentiation control elements (DICE) in Lox mRNA and the pyrimidine-rich motifs in alpha-globin mRNA. In the present report a set of mRNA translation and stability assays are used to determine how these two alphaCP-containing complexes, related in structure and position, mediate distinct posttranscriptional controls. While the previously reported translational silencing by the DICE is not evident in our studies, we find that the two determinants mediate similar levels of mRNA stabilization in erythroid cells. In both cases this stabilization is sensitive to interference by a nuclear-restricted alphaCP decoy but not by the same decoy restricted to the cytoplasm. These data support a general role for alphaCPs in stabilizing a subset of erythroid mRNAs. The findings also suggest that initial binding of alphaCP to target mRNAs occurs in the nucleus. Assembly of stabilizing mRNP complexes in the nucleus prior to export may maximize their impact on cytoplasmic events.

3' Untranslated Regions↗
PubMed2000

The inhibitory effect of the 5' untranslated region of muscle acylphosphatase mRNA on protein expression is relieved during cell differentiation.

Previous experiments suggested that the upstream AUG triplet present in the 5' untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5'UTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5'UTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5'UTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5'UTR of the muscle acylphosphatase mRNA.

5' Untranslated Regions↗
PubMed1996

Positive regulation of corneal type V collagen mRNA: analysis by chicken-human heterokaryon formation.

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for alpha1(V) chain mRNA, and this effect is at least somewhat cell specific.

Animals↗