scholarly journals cDNA sequence, deduced amino acid sequence, predicted gene structure and chemical regulation of mouse Cyp2e1

1992 ◽  
Vol 281 (3) ◽  
pp. 689-695 ◽  
Author(s):  
J E Freeman ◽  
D Stirling ◽  
A L Russell ◽  
C R Wolf
Keyword(s):  
Sequence Similarity ◽  
Mrna Translation ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Coding Region ◽  
Cdna Sequences ◽  
Protein Levels ◽  
Cyp2e1 Gene ◽  
Wide Range ◽  
Dependent Protein

The cDNA encoding the mouse Cyp2e1 protein has been isolated and sequenced, and shown to share 92%, 79%, 80% and 79% sequence similarity over the coding region with rat, human, rabbit 1 and rabbit 2 CYP2E1 cDNA sequences respectively. The predicted Cyp2e1 protein contains 493 amino acids, with a molecular mass of 56781 Da. The protein contains many features common to other cytochrome P450s, including a potentially phosphorylatable serine residue at position 129 within a canonical cyclic AMP-dependent protein kinase site. Southern blot analysis of genomic DNA prepared from C57BL/6 and DBA/2N mice suggests the presence of only a single Cyp2e1 gene. The Cyp2e1 gene was isolated and its organization was established by PCR using oligonucleotides to its predicted intron/exon boundaries. These results showed that the mouse Cyp2e1 gene is approx. 11,000 bp in length and has a similar structure to the human and rat CYP2E1 genes. Cyp2e1 protein expression was studied in a variety of tissues and a sexual dimorphism in its levels in some tissues was noted. Acetone treatment induced the Cyp2e1 protein in all of the tissues studied in both sexes, but this Cyp2e1 protein induction was not accompanied by an increase in Cyp2e1 mRNA levels. Indeed, mRNA levels were seen to be decreased on treatment, suggesting that acetone administration affects either mRNA translation efficiency or protein stability. Of a wide range of drugs known to modify other cytochrome P450 levels only diethylnitrosamine had a significant effect on Cyp2e1, causing a decrease in protein levels.

TPO-Induced miR-150 Down-Regulates c-Myb in Primary Megakaryocytes and a Megakaryocytic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1238-1238 ◽  
Author(s):  
Charlene F. Barroga ◽  
Hang Pham ◽  
Kenneth Kaushansky
Keyword(s):  
Transcription Factor ◽  
Western Blotting ◽  
Luciferase Activity ◽  
Mrna Translation ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Protein Levels ◽  
Micro Rnas

Abstract Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytic progenitors (CFU-MK) and mature megakaryocytes (MK). Thrombopoietin (Tpo), the primary regulator of megakaryopoiesis, induces these same effects, which lead us to hypothesize that Tpo might act, at least in part, through modulation of c-Myb expression. We found using quantitative (Q)-PCR that c-Myb mRNA levels were 13-fold reduced during Tpo-induced MK maturation. Micro RNAs (miRs) are ∼22 nucleotide species that down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs, enhancing mRNA degradation, or by reducing mRNA translation efficiency. We noted that the 3′UTR of c-Myb contains a number of miR target sites, including four that bind miR150; using a specific Q-PCR assay we also found that Tpo increased mir-150 expression to 160% of baseline at 24 hr and 250% at 48 hr in UT7/TPO cells (n=2 experiments). To test if miR150 affects c-Myb expression, we introduced the 3′UTR of c-Myb into a luciferase reporter gene (pCMV-luc-3′UTRcMyb), in which CMV promoter-driven luciferase activity would reflect the stability of the 3′UTR of c-Myb, and allow us to test the effects of miR150 on c-Myb expression in transduced cells; Q-PCR and western blotting were used to simultaneously assess endogenous c-Myb mRNA and protein levels in the cells treated with miR-150 and anti-miR-150, and their respective controls (Ambion, ABI). Co-transfection of UT7/TPO cells with pCMV-luc-3′UTRcMyb and miR-150 significantly down-regulated luciferase activity to 40% of baseline 24 hr following transfection (p = 0.035; n=2 experiments) compared to a miR negative control. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150. Q-PCR analysis revealed that endogenous c-Myb mRNA was significantly down-regulated to 60% of baseline upon transfection of miR-150 compared to the negative control (p = 0.043), while the essential megakaryocytic transcription factor, AML1/RUNX1, remained unaltered. Western blotting of these cell lysates revealed that c-Myb protein expression was down-regulated to 30% of baseline (n=3 experiments) following transduction with miR150 but not with the miR negative control. Converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs, revealed that anti-miR150 significantly upregulated luciferase activity to 180% of baseline compared to an anti-miR-negative control (p=0.003; n=2 experiments). These findings establish that miR-150 down-modulates c-Myb mRNA, and to a greater extent protein levels, suggesting effects on both mRNA stability and protein translation efficiency. And since Tpo affects miR-150 expression, our results also suggest that in addition to direct effects on the survival and growth of MK progenitor cells, mediated by the JAK/STAT, PI3K/Akt and MAPK pathways, Tpo down-modulates c-Myb expression during megakaryopoiesis through the induction of miR150. We are currently ascertaining the in vivo role of miR-150 in Tpo-induced megakaryopoiesis, but these studies already establish that hematopoietic growth factors such as Tpo can influence transcription factor expression through modulation of microRNA species.

scholarly journals Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity

2021 ◽  
Author(s):  
Stephen M Blazie ◽  
Seika Takayanagi-Kiya ◽  
Katherine A McCulloch ◽  
Yishi Jin
Keyword(s):  
Rna Binding ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
Control Activity ◽  
C Elegans ◽  
Protein Levels ◽  
Neuronal Protein

AbstractThe translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of theC. elegansRNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5′UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5′UTR dependent manner. Our study reveals anin vivomechanism for eIF3 in governing neuronal protein levels to control activity states and offers insights into how eIF3 dysregulation contributes to neuronal disorders.

scholarly journals Calcitriol Suppresses HIF-1 and HIF-2 Transcriptional Activity by Reducing HIF-1/2α Protein Levels via a VDR-Independent Mechanism

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2440
Author(s):  
Ioanna-Maria Gkotinakou ◽  
Eleni Kechagia ◽  
Kalliopi Pazaitou-Panayiotou ◽  
Ilias Mylonis ◽  
Panagiotis Liakos ◽  
...  
Keyword(s):  
Vitamin D ◽  
Transcriptional Activity ◽  
Mrna Translation ◽  
Biologically Active ◽  
Mrna Levels ◽  
Akt Signaling Pathway ◽  
Protein Levels ◽  
Anticancer Properties ◽  
Independent Mechanism ◽  
Negative Effect

Hypoxia-inducible transcription factors 1 and 2 (HIFs) are major mediators of cancer development and progression and validated targets for cancer therapy. Although calcitriol, the biologically active metabolite of vitamin D, was attributed with anticancer properties, there is little information on the effect of calcitriol on HIFs and the mechanism underling this activity. Here, we demonstrate the negative effect of calcitriol on HIF-1/2α protein levels and HIF-1/2 transcriptional activity and elucidate the molecular mechanism of calcitriol action. We also reveal that the suppression of vitamin D receptor (VDR) expression by siRNA does not abrogate the negative regulation of HIF-1α and HIF-2α protein levels and HIF-1/2 transcriptional activity by calcitriol, thus testifying that the mechanism of these actions is VDR independent. At the same time, calcitriol significantly reduces the phosphorylation of Akt protein kinase and its downstream targets and suppresses HIF-1/2α protein synthesis by inhibiting HIF1A and EPAS1 (Endothelial PAS domain-containing protein 1) mRNA translation, without affecting their mRNA levels. On the basis of the acquired data, it can be proposed that calcitriol reduces HIF-1α and HIF-2α protein levels and inhibits HIF-1 and HIF-2 transcriptional activity by a VDR-independent, nongenomic mechanism that involves inhibition of PI3K/Akt signaling pathway and suppression of HIF1A and EPAS1 mRNA translation.

scholarly journals Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

2014 ◽  
Vol 13 (5) ◽  
pp. 664-674 ◽  
Author(s):  
Bhaskar Anand Jha ◽  
Abeer Fadda ◽  
Clementine Merce ◽  
Elisha Mugo ◽  
Dorothea Droll ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Mrna Levels ◽  
Coding Region ◽  
Content Type ◽  
Puf Proteins ◽  
Reading Frames

ABSTRACT Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.

Ontogenesis of phase I hepatic drug metabolic enzymes in sheep

2012 ◽  
Vol 24 (3) ◽  
pp. 425 ◽  
Author(s):  
Manoja Pretheeban ◽  
Geoff Hammond ◽  
Stelvio Bandiera ◽  
Wayne Riggs ◽  
Dan Rurak
Keyword(s):  
Similar Data ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Regulatory Factor ◽  
Fetal Sheep ◽  
Hepatic Drug ◽  
Cdna Sequences ◽  
Protein Levels ◽  
Cyp Enzymes ◽  
Rapid Amplification

Cytochrome P450 (CYP) enzymes are important for the metabolism of many drugs. While there is information on their identity and ontogeny in humans and rodents, similar data in sheep are lacking. In the present study, cDNA sequences of several CYP enzymes (CYP2A6, CYP2C19, CYP2D6) were cloned by rapid amplification of cDNA ends. In adult, newborn and fetal sheep the mRNA and protein levels of these CYPs and the regulatory factor, hepatic nuclear factor 4α (HNF4α) were determined in liver samples using real-time PCR and western blotting. The effect of antenatal glucocorticoid on these enzymes was also studied by i.v. infusion of cortisol (0.45 mg h–1; 80 h) to another group of fetuses. The mRNA and protein levels of the CYPs and HNF4α were low or absent in the fetus, followed by increasing levels in the newborn and adult. Fetal cortisol administration significantly increased the mRNA and protein levels of CYP2D6. Moreover, the correlation observed between the CYP and HNF4α mRNA levels suggests a possible regulatory role for this transcription factor. The findings suggest that fetal and newborn lambs have a low ability to metabolise drugs that are substrates of these enzymes, and that this ability increases with advancing postnatal age, similar to the situation in humans.

Control of protein translation by phosphorylation of the mRNA 5′-cap-binding complex

2007 ◽  
Vol 35 (6) ◽  
pp. 1634-1637 ◽  
Author(s):  
O.A. Pierrat ◽  
V. Mikitova ◽  
M.S. Bush ◽  
K.S. Browning ◽  
J.H. Doonan
Keyword(s):  
Environmental Changes ◽  
Mrna Translation ◽  
Cost Effective ◽  
Protein Translation ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Initiation Factors ◽  
Protein Levels ◽  
Translation Initiation Factors ◽  
Cap Binding Complex

Initiation of mRNA translation is a key regulatory step in the control of gene expression. Microarray analysis indicates that total mRNA levels do not always reflect protein levels, since mRNA association with polyribosomes is necessary for protein synthesis. Phosphorylation of translation initiation factors offers a cost-effective and rapid way to adapt to physiological and environmental changes, and there is increasing evidence that many of these factors are subject to multiple regulatory phosphorylation events. The present article focuses on the nature of reversible phosphorylation and the function of the 5′-cap-binding complex in plants.

Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a

1990 ◽  
Vol 10 (4) ◽  
pp. 1476-1483
Author(s):  
R Kaddurah-Daouk ◽  
J W Lillie ◽  
G H Daouk ◽  
M R Green ◽  
R Kingston ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Energy Metabolism ◽  
Sequence Similarity ◽  
Adenovirus Type ◽  
Mrna Levels ◽  
Kinase Gene ◽  
Adenovirus E1a ◽  
Wide Range ◽  
Domain 3 ◽  
Brain Creatine Kinase

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator E1a. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the E1a region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by E1a domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of E1a with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.

Oncogenic NK-Like Homeodomain Proteins Inhibit Etoposide-Induced Apoptosis in T-ALL Cell Lines by Activation of the Polycistronic miR-17-92 Transcript.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2787-2787
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Corinna Meyer ◽  
Hans G. Drexler ◽  
Roderick A.F. MacLeod ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Mrna Translation ◽  
Mature Mirnas ◽  
Mrna Levels ◽  
Homeodomain Proteins ◽  
Aberrant Expression ◽  
Protein Levels ◽  
Micro Rnas

Abstract Homeobox genes of the NK-like familiy, including TLX1, TLX3 and NKX2-5, are ectopically activated in T-cell acute lymphoblastic leukemia (T-ALL) cells mostly via chromosomal aberrations. The pathologic function of these closely related genes is still unclear. Here we analyzed their effect on the C13ORF25 gene, containing the miR-17-92 cluster. Micro RNAs (miRNAs) are a class of small non-coding RNAs which are part of an evolutionarily highly conserved intracellular mechanism, regulating gene expression by hybridization to complementary sequences usually located in the 3′untranslated region of coding transcripts. The primary transcripts (pri-mRNA) are processed to short mature miRNAs, mediating either inhibition of mRNA translation or mRNA cleavage. Aberrant expression of specific miRNAs is involved in oncogenesis as recently described for several human malignancies. The miR-17-92 polycistron encodes miRNAs which decrease E2F1 protein expression. Transcription of both E2F1 and miR-17-92 is induced by MYC, itself a target of E2F1, generating a highly regulated interactive network. Depending on the cellular context, E2F1 performs conflicting tasks by triggering proliferation or inducing apoptosis. We investigated the expression of the miR-17-92 cluster in T-ALL cell lines. Real-time RT-PCR analysis of both miR-17-92 pri-mRNA and mature miRNAs revealed different expression levels in these cells, suggesting a possible implication of the NK-like homeodomain proteins in the regulation of the miR-17-92 cluster in T-ALL. HELA cells transfected with TLX1 or NKX2-5 expression constructs showed elevated miR-17-92 pri-mRNA expression, demonstrating an activating effect. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently showed increased miR-17-92 pri-mRNA levels and decreased E2F1 protein amounts. For functional analysis of these downstream targets, another T-ALL cell line (PEER) was lentivirally transduced with expression constructs for either miR-17-92 or E2F1, resulting in reduced or elevated E2F1 protein levels, respectively. Overexpression of miR-17-92 or E2F1 did not significantly influenced the cell proliferation. However, induction of apoptosis by treating these cells with etoposide, an inhibitor of topoisomerase II, indicated that overexpression of miR-17-92 and E2F1 resulted in enhanced and reduced cell viability, respectively, as analyzed by MTT assay. In summary, these data indicate an activatory effect of oncogenic NK-like homeodomain proteins on miR-17-92 expression, reducing E2F1 protein levels and thereby enhancing survival of leukemic T-cells.

scholarly journals BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites

2006 ◽  
Vol 26 (24) ◽  
pp. 9497-9507 ◽  
Author(s):  
Don Benjamin ◽  
Martin Schmidlin ◽  
Lu Min ◽  
Brigitte Gross ◽  
Christoph Moroni
Keyword(s):  
Protein Kinase ◽  
Mrna Decay ◽  
Protein Kinase B ◽  
Proteasomal Degradation ◽  
Mrna Levels ◽  
Cell Compartment ◽  
Protein Levels ◽  
Dependent Protein

ABSTRACT BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBα−/− cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

scholarly journals noxin, a Novel Stress-Induced Gene Involved in Cell Cycle and Apoptosis

2007 ◽  
Vol 27 (15) ◽  
pp. 5430-5444 ◽  
Author(s):  
Naoki Nakaya ◽  
Jill Hemish ◽  
Peter Krasnov ◽  
Sang-Yong Kim ◽  
Yuri Stasiv ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization Signals ◽  
Protein Levels ◽  
Cell Defense ◽  
Cycle Arrest ◽  
Wide Range ◽  
Dependent Protein ◽  
Response To Stress ◽  
Stress Signals ◽  
Inducible Gene

ABSTRACT We describe a novel stress-induced gene, noxin, and a knockout mouse line with an inactivated noxin gene. The noxin gene does not have sequelogs in the genome and encodes a highly serine-rich protein with predicted phosphorylation sites for ATM, Akt, and DNA-dependent protein kinase kinases; nuclear localization signals; and a Zn finger domain. noxin mRNA and protein levels are under tight control by the cell cycle. noxin, identified as a nitric oxide-inducible gene, is strongly induced by a wide range of stress signals: γ- and UV irradiation, hydrogen peroxide, adriamycin, and cytokines. This induction is dependent on p53. Noxin accumulates in the nucleus in response to stress and, when ectopically expressed, Noxin arrests the cell cycle at G1; although it also induces p53, the cell cycle arrest function of Noxin is independent of p53 activity. noxin knockout mice are viable and fertile; however, they have an enlarged heart, several altered hematopoietic parameters, and a decreased number of spermatids. Importantly, loss or downregulation of Noxin leads to increased cell death. Our results suggest that Noxin may be a component of the cell defense system: it is activated by various stress stimuli, helps cells to withdraw from cycling, and opposes apoptosis.

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