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PubMed2004

Analysis of developing maize plastids reveals two mRNA stability classes correlating with RNA polymerase type.

The plastid genome is transcribed by two distinct RNA polymerases, the PEP encoded by the plastid genome and the NEP encoded in the nucleus. Initial models of plastid transcription held that the NEP is responsible for the transcription of housekeeping genes needed early in development, and that the PEP transcribes genes required for photosynthesis. Recently, this model was challenged by the discovery that all plastid genes are transcribed by NEP in PEP-deficient tobacco plastids, suggesting that mRNA turnover may have a strong role in previously observed transcription patterns. In this study, we provide evidence that the NEP enzyme level decreases as plastids mature. In contrast, production of mRNAs by NEP increases as plastids mature, yet their accumulations remain constant. These results suggest that as plastids mature NEP may become more active, and that mRNA turnover varies between transcripts synthesized by NEP and PEP.

DNA-Directed RNA Polymerases↗

PubMed1994

Influence of transcriptional regulation and mRNA stability on hemopexin gene expression in regenerating liver.

The hepatic response to systemic injury is characterized by alterations in the synthesis of plasma proteins, while acute injury to the liver can lead to rapid proliferation of hepatocytes. The hemopexin gene was found to be markedly induced in rat liver following both sham surgery (SS) and 70% partial hepatectomy (PH), models of systemic injury and hepatic proliferation, respectively. Transcriptional and post-transcriptional regulation of this gene was evaluated to examine the mechanisms of hemopexin mRNA expression in these models. Significant transcriptional activation was observed within 6 h of either surgery, with a more pronounced effect after PH. In both processes, transcription rates returned to baseline values by 24 h after surgery, although marked elevations in mRNA steady-state levels were noted for at least 72 h. At each time point, levels of hemopexin mRNA were more abundant following PH than after SS, in part due to greater transcriptional induction. In addition, posttranscriptional mechanisms appeared to contribute to the increased expression of hemopexin post-PH. The in vivo half-life of the 1.6-kb hemopexin transcript was determined to be considerably greater than 12 h in control, sham-operated, and PH animals. The exceptionally long mRNA half-life appears to be an important but complex factor in the kinetics of hemopexin gene regulation.

Animals↗

PubMed1991

Constitutive production of granulocyte colony-stimulating factor and interleukin-6 by a human lung cancer cell line, KSNY: gene amplification and increased mRNA stability.

A human lung squamous cell carcinoma cell line designated KSNY was established from a patient suffering from marked neutrophilia and polyclonal hyper-gamma-globulinemia. In our previous report, we demonstrated colony-stimulating activities in the culture supernatant of this cell line. To determine the exact molecules for the activities, we investigated the gene expression of various cytokines in KSNY cells and showed the mRNA expression of both granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). We also detected substantial amounts of G-CSF and IL-6 in the culture supernatant with sensitive enzyme-linked immunosorbent assays (ELISA). The amplification of the gene locus for G-CSF, but not for IL-6 was shown by Southern blot analysis. Furthermore, we also showed that the mRNAs for G-CSF and IL-6 were relatively stable in KSNY cells. These findings are thought to be related to the constitutive production of the cytokines in KSNY cells.

Blotting, Northern↗

PubMed1996

B-myb transcriptional regulation and mRNA stability during differentiation of neuroblastoma cells.

B-myb and c-myb expression is high in neuroblastoma cells and declines during retinoic acid-induced differentiation. We show here that B-myb down-regulation during retinoic acid-induced differentiation of LAN-5 neuroblastoma cells occurs at the transcriptional level. In addition, we measured B-myb and c-myb messenger RNA half-lives, and found that, unlike c-myb, B-myb messenger RNA was remarkably stable (> 10 h). Inhibition of protein synthesis by treatment with cycloheximide increased B-myb messenger RNA levels, suggesting that one or more labile proteins act as repressors of B-myb transcription. In the same cell line, blocking protein synthesis decreased the level of c-myb mRNA under both normal and differentiative conditions. Thus, B-myb and c-myb undergo similar transcriptional regulation, but there are specific differences in the stability of their messenger RNAs and in the mechanisms through which their transcription is controlled. These differences could reflect different functional roles played by c-myb and B-myb in neuroblastoma cells.

Cell Cycle Proteins↗

PubMed2006

AUF1 cell cycle variations define genomic DNA methylation by regulation of DNMT1 mRNA stability.

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogeneous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3' untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.

3' Untranslated Regions↗

PubMed2003

Increased interleukin-10 mRNA stability in melanoma cells is associated with decreased levels of A + U-rich element binding factor AUF1.

Abnormal production of interleukin-10 (IL-10) is observed in some pathologic conditions. For example, compared with normal melanocytes, IL-10 expression is elevated in melanoma cells. IL-10 overexpression could inhibit both immune surveillance and tumor rejection. We investigated a potential posttranscriptional mechanism for IL-10 overexpression in melanoma cells. In normal melanocytes, the half-life of IL-10 mRNA is 7 min, whereas in the melanoma cell line MNT1, the half-life is 75 min. This 10-fold difference could account, at least in part, for IL-10 overexpression in MNT1 cells. Examination of the 3'-untranslated region (3'-UTR) of IL-10 mRNA revealed a suspected A + U-rich element (ARE) that might target the mRNA for rapid degradation. Transfection experiments confirmed that these sequences promote rapid degradation when inserted into a normally stable mRNA, indicating ARE functionality. As AREs act via their interactions with ARE-binding proteins, we examined cytoplasmic proteins from normal melanocytes and MNT1 cells for IL-10 ARE-binding activity. Compared with cytoplasmic extracts of normal melanocytes, cytoplasmic extracts of MNT1 cells possess substantially less ARE-binding activity, consistent with the extended half-life of IL-10 mRNA in MNT1 cells. Finally, we find that the ARE-binding protein AUF1 comprises the major ARE-binding activity in cytoplasmic extracts of normal melanocytes. By contrast, AUF1 is not detectable in cytoplasmic extracts of MNT1 cells but appears restricted to the nuclear fraction. Together, these data suggest a mechanism whereby reduced cytoplasmic levels of AUF1 in MNT1 melanoma cells may lead to IL-10 overexpression, with deleterious consequences for tumor surveillance and rejection.

3' Untranslated Regions↗

PubMed1993

Molecular mechanisms regulating the production of collagenase and TIMP in U937 cells: evidence for involvement of delayed transcriptional activation and enhanced mRNA stability.

We have used the human promonocyte-like U937 cell line as a model to study the regulation of interstitial collagenase and tissue inhibitor of metalloproteinases (TIMP) during mononuclear phagocyte development. Our results show that differentiation of U937 cells with exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) induces a temporally delayed (16-24 h) but marked increase in the biosynthesis and secretion of interstitial collagenase and TIMP. Similarly, steady-state mRNA levels for both proteins rose dramatically during the period of exposure, but again after considerable time delay (12-16 h). For interstitial collagenase, induction was transcriptionally regulated as demonstrated by nuclear run-on experiments, and required the synthesis of proteins as indicated by cycloheximide treatment. However, transcriptional activation of collagenase was never observed prior to 10-12 h; since c-fos is rapidly induced in U937 cells and largely disappears by 2 h (Mitchell et al., 1985), our data strongly suggest that collagenase induction in this system cannot be explained simply or entirely by an AP-1-dependent mechanism. Although TIMP steady-state mRNA levels also increased substantially with cellular differentiation, no transcription was detected by run-on experiments. However, TPA exposure markedly prolonged the half-life of TIMP mRNA from 4 h to > 20 h. While cycloheximide treatment completely blocked TPA-mediated induction of collagenase mRNA, it only marginally interfered with simultaneously induced TIMP mRNA levels. Our results demonstrate that differentiation of U937 monocytic cells is accompanied by markedly enhanced production of both interstitial collagenase and TIMP. However, there are multiple, and perhaps differing, molecular mechanisms regulating these responses.

Adult↗

PubMed1996

Regulation of phosphatidylcholine synthesis in maturing type II cells: increased mRNA stability of CTP:phosphocholine cytidylyltransferase.

Pulmonary surfactant phosphatidylcholine synthesis increases in fetal lung type II cells with advancing gestation. This increase is accompanied by an increase in gene and protein expression of CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15), which catalyses a regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. In the present study we investigated the role of transcriptional and post-transcriptional mechanisms in the developmental induction of CT mRNA in maturing type II cells. We found that CT mRNA increased 2-fold from days 18 to 21 of fetal rat gestation (term 22 d). This increase in CT mRNA was not accompanied by a developmental increase in CT gene transcription. However, CT mRNA was more stable on day 21 (t1/2 48 h) compared with that on day 18 (t1/2 17 h). Glucocorticoids have been shown to enhance surfactant phosphatidylcholine synthesis in fetal type II cells. Therefore we also examined the effect of maternal glucocorticoid administration to pregnant rats at 19 d of gestation on CT mRNA expression in fetal type II cells isolated 24 h later. Glucocorticoid treatment did not increase type II cell CT mRNA. As reported previously, however, glucocorticoids increased CT activity in the microsomal membrane fraction of fetal type II cells, whereas no differences in cytosolic CT activity were observed. We conclude that the developmental increase in CT mRNA in fetal type II cells is due to a decreased breakdown of the CT transcript and that glucocorticoids regulate fetal type II cell CT activity at a post-translational level.

Animals↗

PubMed2001

mRNA stability and the secretion signal of HrpA, a pilin secreted by the type III system in Pseudomonas syringae.

Gram-negative bacteria that are pathogenic for animals or plants utilise a specialised Type III secretion system to inject effector proteins into their eukaryotic target cells. The basis for selection of the proteins to be translocated via type III systems is still enigmatic. No clearly defined consensus amino acid sequence that could serve as a specific secretion signal has been identified, and the hypothesis that an mRNA secondary structure acts as the signal has several shortcomings. We have localised a secretion signal that is sufficient to ensure the secretion of the pilin HrpA, a substrate and an indispensable extracellular component of the type III secretion machinery of Pseudomonas syringae pv. tomato DC3000, to the first 15 codons. Transcription of hrpA starts at a single site 42 bp upstream of the first codon. Gene swapping experiments revealed that altering the continuity of the 5' non-translated leader with the region including the secretion signal radically decreased accumulation of the hrpA transcript. These results indicate that an mRNA secondary structure, possibly formed in this region, is important for efficient expression of the gene. The proposed secondary structure is not, however, indispensable for the secretion of HrpA and it does not couple secretion and translation.

5' Flanking Region↗

PubMed1997

Lithium increases melanin-concentrating hormone mRNA stability and inhibits tyrosine hydroxylase gene expression in PC12 cells.

Melanin-concentrating hormone (MCH) is a cyclic peptide involved in the regulation of food-intake behaviour and stress response in mammals. Expression of the MCH gene predominates in hypothalamic neurons. Mechanisms governing the regulation of expression of MCH gene in established cell lines were not explored yet. Here, we analysed the actions of nerve growth factor (NGF), dexamethasone, forskolin and lithium on MCH mRNA levels in the PC12 pheochromocytoma cell line. We compared them with those observed on tyrosine hydroxylase (TH) mRNA, constitutively expressed in PC12 cells, and neurotensin (NT) mRNA, taken as a control. In untreated cells, MCH RNA species of high molecular weight were found. Exposure of cells at a combination of NGF and lithium resulted in decreased expression of these MCH RNAs and in the transient production of mature MCH mRNA. Strikingly, after short exposure of PC12 cells to NGF, lithium per se elicited a marked increase in MCH mRNA levels whilst it exerted a potent inhibitory action on TH mRNA expression. Detailed investigations revealed that lithium enhanced MCH mRNA expression through post-transcriptional mechanisms whereas it regulated TH gene expression mainly at the level of transcription. These results demonstrate that lithium, an agent widely used for treatment of manic depressive illness, can exert an opposite effect on MCH and TH mRNA production in PC12 cells. The MCH gene system in NGF-treated PC12 cells provides a good opportunity for studying the effect of lithium on gene expression at post-transcriptional levels in a neuron-like cellular model.

Adrenal Gland Neoplasms↗

PubMed2025

Restraint of inflammasome-driven cytokine responses through the mRNA stability protein TTP.

Activation of the NLRP3 inflammasome causes extensive disturbance of cellular homeostasis, with Golgi disruption, mitochondrial dysfunction, and changes in intracellular ion concentration occurring rapidly upon stimulation. Given this, it would seem near certain that these changes might also globally affect cellular signaling pathways, yet few, if any, studies have explored this possibility. Here, we combine genomics and phosphoproteomics to identify inhibition of the ERK1/2 MAP kinase signaling cascade upon inflammasome stimulation. This loss of ERK1/2 activity results in rapid inactivation of the mRNA decay-promoting protein tristetraprolin (TTP), with loss of TTP promoting subsequent increased release of cytokines upon pyroptosis. Further, we observe significantly increased levels of TTP expression in patients with inflammatory bowel disease, a disease for which altered cytokine expression is a key driver of pathogenesis. Inflammasome activation thus rapidly inactivates a pathway designed to suppress cytokine release, potentially exacerbating hyperinflammatory states, including those involved in autoinflammatory disease.

Inflammasomes↗

PubMed2006

JNK activation decreases PP2A regulatory subunit B56alpha expression and mRNA stability and increases AUF1 expression in cardiomyocytes.

A central feature of heart disease is a molecular remodeling of signaling pathways in cardiac myocytes. This study focused on novel molecular elements of MAPK-mediated alterations in the pattern of gene expression of the protein phosphatase 2A (PP2A). In an established model of sustained JNK activation, a 70% decrease in expression of the targeting subunit of PP2A, B56alpha, was observed in either neonatal or adult cardiomyocytes. This loss in protein abundance was accompanied by a decrease of 69% in B56alpha mRNA steady-state levels. Given that the 3'-untranslated region of this transcript contains adenylate-uridylate-rich elements known to regulate mRNA degradation, experiments explored the notion that instability of B56alpha mRNA accounts for the response. mRNA time-course analyses with real-time PCR methods showed that B56alpha transcript was transformed from a stable (no significant decay over 1 h) to a labile form that rapidly degraded within minutes. These results were supported by complementary experiments that revealed that the RNA-binding protein AUF1, known to destabilize target mRNA, was increased fourfold in JNK-activated cells. A variety of other stress-related stimuli, such as p38 MAPK activation and phorbol ester, upregulated AUF1 expression in cultured cardiac cells as well. In addition, gel mobility shift assays demonstrated that p37AUF1 binds with nanomolar affinity to segments of the B56alpha 3'-untranslated region. Thus these studies provide new evidence that signaling-induced mRNA instability is an important mechanism that underlies the changes in the pattern of gene expression evoked by stress-activated pathways in cardiac cells.

3' Untranslated Regions↗

PubMed2002

A 3'-UTR variant of the inducible porcine hsp70.2 gene affects mRNA stability.

Large individual differences were observed in the abundance of transcripts from the hsp70.2 gene in primary fibroblast cultures sampled from 15 different pigs. While previously described functional promoter variants of this gene can partly account for the high variability of heat-induced increased abundance of transcripts, they are unrelated to the observed highly variable absolute amounts of hsp70.2 transcripts. Comparative sequence analysis revealed an alteration of the 3'-untranslated region (3'-UTR) sequences in these samples. The variant 3'-UTR allele proved to increase the half life of the hsp70.2 mRNA in reporter gene assays. It is suggested that the cellular stress response is significantly affected by the action and interaction of both promoter and 3'-UTR variants of the hsp70.2 gene.

3' Untranslated Regions↗

PubMed1998

Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase.

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

ADP Ribose Transferases↗

PubMed2004

A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene.

We have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2-3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5-1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to "mutation" or "variation" of pseudogenes.

Animals↗

PubMed2001

Regulation of GP63 mRNA stability in promastigotes of virulent and attenuated Leishmania chagasi.

GP63 is a 63-kDa glycoprotein that is abundantly expressed on the surface of all Leishmania species and is involved in several steps of promastigote infection of host cells. Leishmania chagasi has at least 18 haploid msp (major surface protease) genes encoding GP63 that are divided into three classes, mspS, mspL or mspC, according to their unique 3' UTR sequences and differential expression. All three msp classes are constitutively transcribed during virulent promastigote growth in vitro, although mspL mRNA is most abundant during logarithmic phase and mspS mRNA predominates in stationary phase. Thus, the steady state levels of the mspL and mspS mRNAs are post-transcriptionally regulated. Using Actinomycin D to arrest transcription, we found that in virulent promastigotes the half-life (t(1/2)) of mspL mRNA is coordinately modulated with growth phase, decreasing from a mean of 84 min during early logarithmic growth to a mean of 17 min at a stage intermediate between logarithmic and stationary phase. However, in attenuated promastigotes, the t(1/2) of mspL RNA remains the same throughout parasite growth. In contrast to mspL RNA, the t(1/2) of mspS and mspC RNA is constant throughout all growth phases of both virulent and attenuated promastigote growth. The presence of the translation inhibitor cycloheximide increases the t(1/2) of mspL RNA 4-6-fold in both virulent and attenuated promastigotes at all growth phases. These results indicate that the t(1/2) of mspL RNA is maintained by at least two distinct mechanisms - one activated during growth to stationary phase and the other dependent on a labile negative regulatory protein factor(s).

Animals↗

PubMed2002

Sarcoplasmic reticulum Ca(2+) pump mRNA stability in cardiac and smooth muscle: role of the 3'-untranslated region.

Stomach smooth muscle (SSM) and left ventricular muscle (LVM) express the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump gene SERCA2. Alternative splicing yields two major isoforms, SERCA2a in LVM and slow twitch muscle and SERCA2b in SSM and most other tissues. The splices have different 3'-untranslated regions (UTR) and also encode proteins that differ slightly in their COOH-terminal domains. SERCA2 transcription rates are similar in the two tissues, yet LVM has a much higher level of SERCA2 mRNA than SSM. To understand the control of SERCA2 RNA expression, we inhibited transcription and showed that the half-life of SERCA2 mRNA is significantly longer (P < 0.05) in primary cultures of LVM cells than in SSM cells. Nuclear SERCA2 mRNA levels were also higher in LVM than in SSM. In vitro decay assays using synthetic RNA corresponding to the 3'-UTR of SERCA2a and -2b showed that nuclear extracts produced a faster decay of SERCA2 RNA than cytoplasmic extracts and that nuclear extracts produced a faster decay of SERCA2b than -2a. This was also true when the full-length native mRNA was used instead of the 3'-UTR RNA, and SERCA2b decay by cytoplasmic extracts was faster for LVM than for SSM. We propose that nuclear decay is an initial step in the control of SERCA2 RNA abundance and that this control is maintained or modulated in the cytoplasm. We discuss how these control mechanisms may be part of a control switch in cardiac development and pathophysiology.

3' Untranslated Regions↗

PubMed2002

Effects on collagen VI mRNA stability and microfibrillar assembly of three COL6A2 mutations in two families with Ullrich congenital muscular dystrophy.

We recently reported a severe deficiency in collagen type VI, resulting from recessive mutations of the COL6A2 gene, in patients with Ullrich congenital muscular dystrophy. Their parents, who are all carriers of one mutant allele, are unaffected, although heterozygous mutations in collagen VI caused Bethlem myopathy. Here we investigated the consequences of three COL6A2 mutations in fibroblasts from patients and their parents in two Ullrich families. All three mutations lead to nonsense-mediated mRNA decay. However, very low levels of undegraded mutant mRNA remained in patient B with compound heterozygous mutations at the distal part of the triple-helical domain, resulting in deposition of abnormal microfibrils that cannot form extensive networks. This observation suggests that the C-terminal globular domain is not essential for triple-helix formation but is critical for microfibrillar assembly. In all parents, the COL6A2 mRNA levels are reduced to 57-73% of the control, but long term collagen VI matrix depositions are comparable with that of the control. The almost complete absence of abnormal protein and near-normal accumulation of microfibrils in the parents may account for their lack of myopathic symptoms.

Alleles↗