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Eukaryotic Cells↗
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Identifying transcribed sequences, and beyond.
A report on the 12th International Workshop 'Beyond the Identification of Transcribed Sequences (BITS): Functional, Expression and Evolutionary Analysis', Washington DC, USA, 25-28 October 2002.
Alternative Splicing↗
An unappreciated role for RNA surveillance.
BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons (PTC+ mRNAs). In mammals, a termination codon is recognized as premature if it lies more than about 50 nucleotides upstream of the final intron position. More than a third of reliably inferred alternative splicing events in humans have been shown to result in PTC+ mRNA isoforms. As the mechanistic details of NMD have only recently been elucidated, we hypothesized that many PTC+ isoforms may have been cloned, characterized and deposited in the public databases, even though they would be targeted for degradation in vivo. RESULTS: We analyzed the human alternative protein isoforms described in the SWISS-PROT database and found that 144 (5.8% of 2,483) isoform sequences amenable to analysis, from 107 (7.9% of 1,363) SWISS-PROT entries, derive from PTC+ mRNA. CONCLUSIONS: For several of the PTC+ isoforms we identified, existing experimental evidence can be reinterpreted and is consistent with the action of NMD to degrade the transcripts. Several genes with mRNA isoforms that we identified as PTC+--calpain-10, the CDC-like kinases (CLKs) and LARD--show how previous experimental results may be understood in light of NMD.
Alternative Splicing↗
Natural antisense transcripts with coding capacity in Arabidopsis may have a regulatory role that is not linked to double-stranded RNA degradation.
BACKGROUND: Overlapping transcripts in antisense orientation have the potential to form double-stranded RNA (dsRNA), a substrate for a number of different RNA-modification pathways. One prominent route for dsRNA is its breakdown by Dicer enzyme complexes into small RNAs, a pathway that is widely exploited by RNA interference technology to inactivate defined genes in transgenic lines. The significance of this pathway for endogenous gene regulation remains unclear. RESULTS: We have examined transcription data for overlapping gene pairs in Arabidopsis thaliana. On the basis of an analysis of transcripts with coding regions, we find the majority of overlapping gene pairs to be convergently overlapping pairs (COPs), with the potential for dsRNA formation. In all tissues, COP transcripts are present at a higher frequency compared to the overall gene pool. The probability that both the sense and antisense copy of a COP are co-transcribed matches the theoretical value for coexpression under the assumption that the expression of one partner does not affect the expression of the other. Among COPs, we observe an over-representation of spliced (intron-containing) genes (90%) and of genes with alternatively spliced transcripts. For loci where antisense transcripts overlap with sense transcript introns, we also find a significant bias in favor of alternative splicing and variation of polyadenylation. CONCLUSION: The results argue against a predominant RNA degradation effect induced by dsRNA formation. Instead, our data support alternative roles for dsRNAs. They suggest that at least for a subgroup of COPs, antisense expression may induce alternative splicing or polyadenylation.
Alternative Splicing↗
Pro- and anti-inflammatory substances modulate expression of the leukotriene B4 receptor, BLT1, in human monocytes.
The high-affinity leukotriene B(4) (LTB(4)) receptor, BLT1, is a chemotactic receptor involved in inflammatory responses. In this study, we have explored the regulation of BLT1 expression in human monocytes by pro- and anti-inflammatory cytokines, lipopolysaccharide (LPS), and dexamethasone. We found that proinflammatory mediators, such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha, and LPS, down-regulated expression, whereas the anti-inflammatory cytokine, interleukin-10, and dexamethasone up-regulated BLT1 mRNA expression. The effect of IFN-gamma on BLT1 mRNA expression was rapidly detectable (<4 h) and concentration-dependent (1-50 ng/ml) and seems to be exerted through a block in transcriptional activity. Alterations in mRNA expression were accompanied by changes in BLT1 surface expression, and receptor down-modulation following IFN-gamma stimulation resulted in a diminished chemotactic response to LTB(4). The regulation of BLT1 mRNA and receptor protein expression was similar to the regulation of the monocyte chemoattractant protein-1 chemokine receptor, CC chemokine recptor 2 (CCR2). Flow cytometric analysis of fresh peripheral blood cells revealed that classical (CD14(++)CD16(-)) monocytes express high levels of BLT1 and CCR2 and that both receptors are down-regulated on CD14(+)CD16(+) monocytes. Apart from providing insight into the regulation of BLT1 in human monocytes, our results reveal a parallel expression and regulation of BLT1 and CCR2, which may help to understand monocyte trafficking during pathophysiological conditions.
Chemotaxis↗
A nucleotide insertion in the transcriptional regulatory region of FADS2 gives rise to human fatty acid delta-6-desaturase deficiency.
Fatty acid delta-6-desaturase (FADS2) is the rate-limiting enzyme in mammalian synthesis of long-chain polyunsaturated fatty acids. We investigated the molecular mechanism of FADS2 deficiency in skin fibroblasts from a patient deficient in this enzyme. Expression analyses demonstrated an 80% to 90% decrease in the steady-state level of FADS2 mRNA in patient-derived cells compared with normal controls that was consistent with previous metabolic biochemical studies. In vitro transcription assays indicated an 80% decrease in the rate of transcriptional initiation in patient-derived cells, thus implicating transcriptional regulation as the mechanism for the decreased transcript levels. Sequence analysis of the 5' end of the gene revealed the insertion of a thymidine between positions -941 and -942 upstream of the translation start site in patient-derived cells compared with normal cells and published sequences. Promoter-reporter assays demonstrated a 6-fold decrease in promoter activity in the polymorphic variant FADS2 regulatory region compared with the normal gene, confirming the functional relevance of the insertion mutation to the decreased expression of the gene in the patient-derived cells. These findings indicate that fatty acid delta-6-desaturase deficiency and decreased FADS2 transcription are caused by a nucleotide insertion in the transcriptional regulatory region of the human FADS2 gene.
3T3 Cells↗
PPARalpha controls the intracellular coenzyme A concentration via regulation of PANK1alpha gene expression.
Pantothenate kinase (PanK) is thought to catalyze the first rate-limiting step in CoA biosynthesis. The full-length cDNA encoding the human PanK1alpha protein was isolated, and the complete human PANK1 gene structure was determined. Bezafibrate (BF), a hypolipidemic drug and a peroxisome proliferator activator receptor-alpha (PPARalpha) agonist, specifically increased hPANK1alpha mRNA expression in human hepatoblastoma (HepG2) cells as a function of time and dose of the drug, compared with hPANK1beta, hPANK2, and hPANK3, which did not significantly increase. Four putative PPARalpha response elements were identified in the PANKIalpha promoter, and BF stimulated hPANK1alpha promoter activity but did not alter the mRNA half-life. Increased hPANK1alpha mRNA resulted in higher hPanK1 protein, localized in the cytoplasm, and elevated PanK enzyme activity. The enhanced hPANK1alpha gene expression translated into increased activity of the CoA biosynthetic pathway and established a higher steady-state CoA level in HepG2 cells. These data are consistent with a key role for PanK1alpha in the control of cellular CoA content and point to the PPARalpha transcription factor as a major factor governing hepatic CoA levels by specific modulation of PANK1alpha gene expression.
Amino Acid Sequence↗
n-6 PUFAs downregulate expression of the tricarboxylate carrier in rat liver by transcriptional and posttranscriptional mechanisms.
The tricarboxylate (citrate) carrier (TCC), a protein of the mitochondrial inner membrane, is an obligatory component of the shuttle system by which mitochondrial acetyl-CoA is transported into the cytosol, where lipogenesis occurs. The aim of this study was to investigate the molecular basis for the regulation of TCC gene expression by a high-fat, n-6 PUFA-enriched diet. Rats received for up to 4 weeks a diet enriched with 15% safflower oil (SO), which is high in linoleic acid (70.4%). We found a gradual decrease of TCC activity and a parallel decline in the abundance of TCC mRNA, the maximum effect occurring after 4 weeks of treatment. At this time, the estimated half-life of TCC mRNA was the same in the hepatocytes from rats on both diets, whereas the transcriptional rate of TCC mRNA, tested by nuclear run-on assay, was reduced by approximately 38% in the rats on the SO-enriched diet. The RNase protection assay showed that the ratio of mature to precursor RNA, measured in the nuclei, decreased with the change to the n-6 PUFA diet. These results suggest that administration of n-6 PUFAs to rats leads to changes not only in the transcriptional rate of the TCC gene but also in the processing of the nuclear precursor for TCC RNA.
Animals↗
Resistin expression in 3T3-L1 adipocytes is reduced by arachidonic acid.
The resistin gene is expressed in adipocytes and encodes a protein proposed to link obesity and type 2 diabetes. Increased plasma FFA is associated with insulin resistance. We examined the effect of separate FFAs on the expression of resistin mRNA in cultured murine 3T3-L1 adipocytes. The FFAs tested did not increase resistin expression, whereas both arachidonic acid (AA) and eicosapentaenoic acid (EPA) reduced resistin mRNA levels. AA was by far the most potent FFA, reducing resistin mRNA levels to approximately 20% of control at 60-250 muM concentration. Selective inhibitors of cyclooxygenase-1 and of mitogen-activated protein kinase kinase counteracted AA-induced reduction in resistin mRNA levels. Transient overexpression of sterol-regulatory element binding protein-1a (SREBP-1a) activated the resistin promoter, but there was no reduction in the abundance of approximately 65 kDa mature SREBP-1 after AA exposure. Actinomycin D as well as cycloheximide abolished the AA-induced reduction of resistin mRNA levels, indicating dependence on de novo transcription and translation. Our data suggest that reductions in resistin mRNA levels involve a destabilization of the resistin mRNA molecule. An inhibitory effect of AA and EPA on resistin expression may explain the beneficial effect of ingesting PUFAs on insulin sensitivity.
3T3-L1 Cells↗
LXR and PPAR activators stimulate cholesterol sulfotransferase type 2 isoform 1b in human keratinocytes.
Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are potent regulators of keratinocyte proliferation, differentiation, and epidermal permeability barrier homeostasis. Cholesterol sulfotransferase type 2B isoform 1b (SULT2B1b) is a key enzyme in the synthesis of cholesterol sulfate (CS), a critical regulator of keratinocyte differentiation and desquamation, as well as a mediator of barrier homeostasis. In this study, we assessed the effect of activators of LXR, PPARalpha, PPARbeta/delta, and PPARgamma on SULT2B1b gene expression and enzyme activity in cultured human keratinocytes (CHKs). Our results demonstrate that PPAR and LXR activators increase SULT2B1b mRNA levels, with the most dramatic effect (a 26-fold increase) induced by the PPARgamma activator ciglitazone. Ciglitazone upregulates SULT2B1b mRNA in a dose- and time-dependent manner. Moreover, the stimulation of SULT2B1b gene expression by LXR and PPAR activators occurs in both undifferentiated and differentiated CHKs. The upregulation of SULT2B1b mRNA by ciglitazone appears to occur at a transcriptional level, because the degradation of SULT2B1b is not accelerated by ciglitazone. In addition, cycloheximide almost completely blocks the ciglitazone-induced increase in SULT2B1b mRNA, suggesting that the transcription of SULTB1b mRNA is dependent on new protein synthesis. Finally, LXR and PPAR activators also increased the activity of cholesterol sulfotransferase. Thus, LXR and PPAR activators regulate the expression of SULT2B1b, the key enzyme in the synthesis of CS, which is a potent regulator of epidermal differentiation and corneocyte desquamation.
Cell Differentiation↗
The human placental lactogen genes: structure, function, evolution and transcriptional regulation.
hPL is a member of an evolutionarily related gene family including hGH and hPRL. Expression of hPL is limited to the placenta but its physiological actions are far reaching. hPL has a direct somatotropic effect on fetal tissues, it alters maternal carbohydrate and lipid metabolism to provide for fetal nutrient requirements, and aids in stimulation of mammary cell proliferation. Two hPL genes (hPL3 and hPL4) encoding identical proteins are responsible for the production of up to 1-3 g PL hormone/day. Recent studies have characterized the regulatory controls of hPL expression. At the post transcriptional level, RNA stability may contribute to variable levels of hPL3 vs. hPL4 production. In addition, non-tissue-specific protein-promoter interactions involving the Sp1 transcription factor are necessary for hPL transcription initiation. A transcriptional enhancer located 3' to the hPL3 gene is responsible for the placenta-specific expression of this gene, while an additional enhancer may be located 3' to the hPl4 gene. The hPL enhancer is bound by multiple proteins including at least one placental specific protein that interacts with a TEF-1 motif. Therefore, enhancer-protein interactions most likely play a large part in the high levels of placenta-specific hPL expression.
Amino Acid Sequence↗
Differential regulation of two 3' end variants of P450 aromatase transcripts and of a new truncated aromatase protein in rabbit preovulatory granulosa cells.
In rabbit granulosa cells, two cytochrome P450 aromatase (P450 arom) mRNAs issued from promoter II were described: a full-length and a truncated transcript. Western blot analysis showed two P450 arom proteins with apparent molecular masses of 53 and 46 kDa, which are consistent with the predicted theoretical sizes of proteins encoded by these two transcripts. To examine the involvement of the truncated transcript in the regulation of P450 arom gene expression, the level of each transcript was specifically quantified in cultured granulosa cells by competitive quantitative RT-PCR. FSH induced a dose-dependent increase in both estradiol production and P450 arom mRNAs levels with a much more enhancement in the full-length mRNA. The half-life of the transcripts could not explain this differential regulation. Upon dibutyryl cAMP stimulation, the full-length mRNA was less abundant than the truncated one. In contrast, Western blot analysis revealed a stimulation of the 53-kDa protein content, whereas the 46-kDa protein amount was apparently unaffected. TGF beta in FSH-stimulated conditions decreased both estradiol production and P450 arom transcripts levels. TGF beta did not modify estradiol production and aromatase protein amounts induced by dibutyryl cAMP, whereas the two P450 arom mRNAs levels were increased. In conclusion, we report for the first time that a protein encoded by a truncated P450 arom mRNA could be involved in the regulation of estrogen production. Moreover, we show that the two P450 arom mRNAs are regulated in a differential manner, probably through hormonal control of the alternative splicing.
Animals↗
Ghrelin increases neuropeptide Y and agouti-related peptide gene expression in the arcuate nucleus in rat hypothalamic organotypic cultures.
Ghrelin, which was identified from the rat stomach, is a potent stimulant for food intake. Several lines of evidence suggest that the orexigenic action of ghrelin is mediated via the neuropeptide Y (NPY) neurons in the arcuate nucleus, although the detailed mechanisms by which ghrelin stimulates NPY neurons are not clear. In this study, we examined the gene regulation of NPY and agouti-related peptide (AGRP), another orexigenic peptide synthesized in the NPY neurons, in the arcuate nucleus by ghrelin in hypothalamic organotypic cultures. Incubation of the hypothalamic explants with ghrelin significantly increased NPY and AGRP mRNA expression in the presence, but not absence, of dexamethasone. Glucocorticoids were also necessary for ghrelin action in vivo because an intracerebroventricular injection of ghrelin significantly increased NPY and AGRP mRNA expression in the arcuate nucleus only in sham-operated, but not in adrenalectomized rats. The stimulatory effects of ghrelin on gene expression were not blocked by a sodium channel blocker tetrodotoxin in the organotypic cultures. Ghrelin also increased NPY heteronuclear (hn) RNA expression, the first transcript that has been used as an indicator for gene transcription. The stimulatory effects of ghrelin on NPY gene expression were abolished in the presence of cycloheximide, which blocks translation, suggesting that de novo protein synthesis is required for ghrelin action. These data suggest that ghrelin stimulates NPY and AGRP gene expression independently of action potentials only in the presence of glucocorticoids. Furthermore, our data demonstrate stimulatory action of ghrelin on NPY gene transcription, which requires de novo protein synthesis.
Agouti-Related Protein↗
The thyrotropin beta-subunit gene is repressed by thyroid hormone in a novel thyrotrope cell line, mouse T alphaT1 cells.
TSH is expressed in two populations of thyrotropes in the pituitary: one in the pars distalis and a second in the pars tuberalis. Pars distalis thyrotropes exhibit classical endocrine inhibition of TSH by thyroid hormone, whereas pars tuberalis thyrotropes do not. The majority of our understanding of TSH subunit gene regulation has come from studies conducted in dispersed pituitary, dispersed thyrotropic tumors, or the GH3 somatolactotrope cell line. However, the dispersed pituitary model is limited because of its inherent heterogeneity, thyrotropic tumors are difficult to grow and maintain, and the GH3 cells lack endogenous TSH expression. The recent derivation of a clonal thyrotrope cell line, T alphaT1, that expresses thyrotrope-specific markers, overcomes these limitations. However, because it was not possible to distinguish whether the tumor from which the T alphaT1 cells are derived originated in the pars distalis or the pars tuberalis, it was necessary to define their cellular origin and thereby establish their status as representative thyrotrope cells for future molecular studies. In this study, we demonstrate that the T alphaT1 cells express thyroid hormone receptors (beta1 and beta2) and their heterodimeric partner, retinoid X receptor-gamma. Treatment with T3 causes a dose- and time-dependent decrease in the expression of the TSH beta-subunit messenger RNA. In contrast to previous reports in rat pituitary cultures, T3 does not alter TSH beta-subunit messenger RNA stability in the T alphaT1 cells. Based on these data and the presence of thyrotrope-specific isoforms of the transcription factor Pit-1, we conclude that the T alphaT1 cells represent differentiated thyrotropes of the pars distalis and will be a useful model system for future analysis of the cis- and trans-acting factors necessary for thyrotrope-specific and thyroid hormone-regulated TSH gene expression.
Animals↗
Differential regulation of IGF-binding protein gene expression by cAMP in rat C6 glioma cells.
We previously reported that cAMP inhibits autocrine IGF-I gene expression in rat C6 glioma cells. In this study we examined the influence of cAMP on IGF-binding protein gene expression in C6 cells. cAMP potently inhibited IGF-binding protein-3 mRNA and, to a lesser extent, IGF-binding protein-4 mRNA and transiently stimulated IGF-binding protein-5 mRNA. The changes in secreted IGF-binding proteins whose molecular weights were consistent with IGF-binding protein-3 and -5 correlated with those of mRNA levels. cAMP decreased the IGF-binding protein-3 mRNA half-life, but did not alter IGF-binding protein-4 and -5 mRNA half-lives. An IGF-binding protein-5 promoter/luciferase fusion construct containing 888 bp of 5'-flanking sequence and the first 114 bp of exon 1 sequence was stimulated by cAMP after 24 h by approximately 2-fold in transient transfection assays. 5'- or 3'-deletion to -33 or +10 (the transcription start site was designated as +1), respectively, did not alter the increase caused by cAMP. Site-directed mutagenesis of the region from -14 to -5 led to a loss of the ability of the IGF-binding protein-5 promoter to respond to cAMP. H89, a cell-permeable protein kinase A inhibitor, did not alter the regulation of IGF-binding protein mRNAs in response to cAMP.
Animals↗
Chicken ovalbumin upstream promoter-transcription factor is a negative regulator of steroidogenesis in bovine adrenal glomerulosa cells.
The octapeptide hormone, angiotensin II (AngII) and ACTH stimulate mineralocorticoid biosynthesis in the zona glomerulosa of the adrenal cortex in part by promoting the transcription of the gene coding for the steroidogenic acute regulatory (StAR) protein. We have examined whether chicken ovalbumin upstream promoter-transcription factor (COUP-TF), a member of the orphan nuclear receptor family of transcription factors, is involved in this transcriptional regulation. We analyzed COUP-TF and StAR mRNA and protein levels in bovine adrenal glomerulosa cells in primary culture. COUP-TF protein was readily detectable in nonstimulated cells, and AngII markedly reduced its expression in a time- and concentration-dependent manner (IC50 = 1 nm), to 46 +/- 4.4% of control levels after 6 h, (n = 3; P < 0.01). This repression was paralleled by a marked decrease in COUP-TF mRNA levels, reaching 18 +/- 8.8% of controls (n = 3, P < 0.01) after 6 h and by a 20-fold increase in aldosterone output. In bovine glomerulosa cells overexpressing COUP-TFI and -II, the induction of StAR mRNA and protein elicited by AngII was completely suppressed to control levels, and the aldosterone response was significantly reduced (from 4.8 +/- 1.1-fold the basal value in mock-infected cells to 1.9 +/- 0.5-fold and 2.2 +/- 0.7-fold in COUP-TFI- and COUP-TFII-expressing cells, respectively; n = 3; P < 0.01 for both differences). Finally, by using electrophoretic mobility shift assays and chromatin immunoprecipitation, we have shown a direct interaction between COUP-TF and the proximal StAR promoter. These results suggest that COUP-TF exerts a tonic inhibition on steroidogenesis by repressing StAR protein expression and that activators of aldosterone biosynthesis lift this inhibition in part by repressing COUP-TF levels.
Aldosterone↗
Messenger RNA degradation may be inhibited in sporadic inclusion body myositis.
OBJECTIVE: To integrate an immune-mediated mechanism and the disturbed protein expression in sporadic inclusion body myositis (IBM). BACKGROUND: In IBM, abnormal fibers harbor inclusions of some proteins found in the brains of patients with Alzheimer disease (AD). Poly(A)-binding protein 1 (PABP1) is the RNA binding protein that attaches to the poly(A) tail of mRNA and is involved in translation and mRNA degradation. Under stresses, mRNA combined with PABP1 forms cytoplasmic granules called stress granules. METHODS: Using 12 muscle biopsies with sporadic IBM and 46 controls, the authors localized PABP1 by immunohistochemistry, and poly(A)-containing RNA (poly(A)+ RNA) using the in situ hybridization method. They also immuno-localized HuR, one of the components of stress granules. RESULTS: In IBM, a proportion of fibers, including those vacuolated, showed an abnormal accumulation of PABP1 immuno-positive deposits. An immunofluorescence study indicated that large PABP1 positive deposits formed conglomerates with poly(A)+ RNA and PABP1 colocalized with HuR. Although PABP1-positive cytoplasmic inclusions were found in disease controls, their aggregates combined with poly(A)+ RNA were only detected in IBM. CONCLUSIONS: The localization of PABP1 positive deposits in inclusion body myositis (IBM) and other diseases may correspond to the stress granules that are formed under exposure to cellular stresses and the sites of mRNA turnover. The concomitant aggregation of poly(A)+ RNA that is specifically found in IBM may be due to the inhibition of mRNA degradation, which may affect translation. The authors speculate that an autoantibody against mRNA degradation machinery could play a role in this inhibition.
Aged↗
Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries.
It is widely believed that translation occurs only in the cytoplasm of eukaryotes, but recent results suggest some takes place in nuclei, coupled to transcription. Support for this heterodoxy comes from studies of the nonsense-mediated decay (NMD) pathway; this pathway probably uses ribosomes to proofread messenger RNAs. We find components of the machineries involved in transcription, translation and NMD colocalise, interact and copurify, and that interactions between them are probably mediated by the C-terminal domain of the catalytic subunit of RNA polymerase II. These results are simply explained if the NMD machinery uses nuclear ribosomes to translate - and so proofread - newly made transcripts; then, faulty transcripts and any truncated peptides produced by nuclear translation would be degraded.
Animals↗