The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1, Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

PubMed Central

Liu, Chang; Wu, Zhe; Sun, Hong-chen

2009-01-01

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups (n=24). Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-β1 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket. PMID:20687301

Identification of Relationships Between Interleukin 15 mRNA and Brain-Derived Neurotrophic Factor II mRNA Levels With Formal Components of Temperament in Asthmatic Patients.

PubMed

Panek, Michał; Jonakowski, Mateusz; Zioło, Jan; Pietras, Tadeusz; Wieteska, Łukasz; Małachowska, Beata; Mokros, Łukasz; Szemraj, Janusz; Kuna, Piotr

2017-04-01

Asthma is a chronic inflammatory and heterogeneous disease developing mostly through allergic inflammation, which modifies the expression of various cytokines and neurotrophins. Previous studies suggest the involvement of interleukin (IL)-15 in the regulation of immune response in asthma. Brain-derived neurotrophic factor (BDNF) II plays an important role as a regulator of development and survival of neurons as well as maintenance of their physiological activity. Chronic stress associated with asthma and elevated IL-15 mRNA and BDNFII mRNA levels may affect the mood and a subjective sensation of dyspnoea-inducing anxiety. Psychopathological variables and numerous cytokine/neurotrophin interactions influence the formation of temperament and strategies of coping with stress. The aim of the study was to identify the role of IL-15 mRNA and BDNFII mRNA expressions and their effect on components of temperament and strategies of coping with stress in asthmatics. A total of 352 subjects (176 healthy volunteers and 176 asthmatic patients) participated in the study. The Formal Characteristic of Behaviour-Temperament Inventory (FCB-TI), Coping Inventory for Stressful Situations (CISS), Beck Depression Inventory, State-Trait Anxiety Inventory, and Borg Rating of Perceived Exertion (RPE) Scale were applied in all the subjects. The expression of IL-15 and BDNFII gene was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Different levels of IL-15 and BDNFII expressions between healthy volunteers and patients were revealed in the study. IL-15 enhanced the BDNFII mRNA expression among patients with bronchial asthma. The depression level negatively correlated with the BDNFII mRNA expression. This neurotrophin modified the temperament variable. BDNFII significantly affected (proportional relationship) the level of briskness in asthmatic patients. BDNFII might influence the level and style of coping with stress (emotion-oriented style). This hypothesis

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

PubMed

Sato, K; Yamazaki, K; Shizume, K; Kanaji, Y; Obara, T; Ohsumi, K; Demura, H; Yamaguchi, S; Shibuya, M

1995-09-01

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

PubMed

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

PubMed

Yan, Xiaodong; Liu, Juanfang; Zhang, Zhengping; Li, Wenhao; Sun, Siguo; Zhao, Jian; Dong, Xin; Qian, Jixian; Sun, Honghui

2017-01-01

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca 2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca 2+ ] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca 2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca 2+ ) stores. Blockade of Ca 2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca 2+ -ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca 2+ -dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

[Influence of macrophages on the expression of vascular endothelial growth factor receptor mRNA, homeobox B2 mRNA, and integrin alpha nu beta3 in vascular endothelial strain].

PubMed

Liu, Liang; Liu, Chang; Zhang, Xiao-qi; Ming, Jia; Liu, Xu-sheng; Xu, Hui; Cheng, Tian-min

2005-06-01

To investigate the influence of macrophages on the expression of the vascular endothelial growth factor (VEGF) receptor (KDR) mRNA, homeobox B2 (HOXB2) mRNA, and integrin alpha nu beta3 in vitro in vascular endothelial strain. Human umbilical vein cells (ECV304) were cultured in vitro and divided into 4 groups, i.e. (1) ECV304 group, (2) ECV304 + conA group [with conA (25 microg/ml in culture) added to ECV304], (3) ECV304 + U937 group (with 1 x 10(5)/ml of U937 cells added to 1 x 10(5)/ml ECV 304), (4) ECV304 + U937 + conA group [with 1 x 10(5)/ml of U937 cells and conA (25 microg/ml in culture)] groups. Forty-eight hours after culturing, the expression of integrin receptor alpha nu beta3 and the changes in the expression of KDR mRNA and HOXB2 mRNA in each group were determined by immunofluorescent technique and RT-PCR, respectively. The expression of integrin receptor alpha nu beta3, KDR mRNA, and HOXB2 mRNA in ECV304 group were 6.7 +/- 1.5, 0.633 +/- 0.012, and 0.674 +/- 0.004, respectively, while those in ECV304 + U937 + conA group (10.2 +/- 1.7, 0.879 +/- 0.003, 0.947 +/- 0.003) were obviously more upregulated when compared with those in ECV304 group (P < 0.01). No difference in the above indices was found between ECV304 and ECV304 + conA, ECV304 + U937 groups (P > 0.05). Macrophages activated by ConA can accelerate the proliferation, migration and adhesion to the basement membrane matrix of vascular endothelial cells through the influence on the expression of KDR mRNA, HOXB2 mRNA and integrin alpha nu beta3, and through this pathway the angiogenesis is modulated.

Prognostic Significance of Human Apurinic/Apyrimidinic Endonuclease (APE/Ref-1) Expression in Rectal Cancer Treated With Preoperative Radiochemotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jun-Sang, E-mail: k423j@cnu.ac.kr; Cancer Research Institute, Chungnam National University, Daejeon; Kim, Jin-Man

Purpose: Human apurinic endonuclease/redox factor 1 (APE/Ref-1) mediates repair of radiation-induced DNA lesions and regulates transcription via redox-based activation. We investigated the predictive and prognostic significance of APE/Ref-1 expression in pretreatment biopsy specimens in locally advanced rectal cancer (LARC) (cT3-T4 or N+). Methods and Materials: APE/Ref-1 expression was analyzed by immunohistochemistry in pretreatment biopsy specimens obtained from 83 patients with LARC. Patients received preoperative radiotherapy of 50.4 Gy in 28 fractions, combined with oral capecitabine and leucovorin chemotherapy, followed by curative surgery. The prognostic significance of various clinicopathologic characteristics, including APE/Ref-1 protein expression, was evaluated. Results: APE/Ref-1 was expressed inmore » 97% of patient samples. Exclusive APE/Ref-1 nuclear staining was observed in 49 of 83 samples (59%), and mixed nuclear and cytoplasmic staining was observed in 31 samples (37%). APE/Ref-1 nuclear expression levels were low in 49 patients (59%) and high in 34 patients (41%). The level of APE/Ref-1 nuclear expression was not a prognostic factor for overall and disease-free survival. Cytoplasmic expression of APE/Ref-1 was a borderline-significant predictive factor for pathologic tumor response (p = 0.08) and a significant prognostic factor for disease-free survival, as shown by univariate analysis (p = 0.037). Multivariate analysis confirmed that cytoplasmic localization of APE/Ref-1 is a significant predictor of disease-free survival (hazard ratio, 0.45; p = 0.046). Conclusions: APE/Ref-1 was expressed in a majority of pretreatment biopsy specimens from patients with LARC. The level of APE/Ref-1 nuclear expression was not a significant predictive and prognostic factor; however, cytoplasmic localization of the protein was negatively associated with disease-free survival. These results indicate that cytoplasmic expression of APE/Ref-1 represents an

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Transcription factor Brn-3α mRNA in cancers, relationship with AR, ER receptors and AKT/m-TOR pathway components

NASA Astrophysics Data System (ADS)

Spirina, L. V.; Gorbunov, A. K.; Chigevskaya, S. Y.; Usynin, Y. A.; Kondakova, I. V.; Slonimskaya, E. M.; Usynin, E. A.; Choinzonov, E. L.; Zaitseva, O. S.

2017-09-01

Transcription factors POU4F1 (neurogenic factor Brn-3α) play a pivotal role in cancers development. The aim of the study was to reveal the Brn-3α expression, AR, ER expression in cancers development, association with AKT/mTOR pathway activation. 30 patients with locally advanced prostate cancer, 20 patients with papillary thyroid cancer, T2-3N0-1M0 stages and 40 patients with renal cell cancer T2-3N0M0-1 were involved into the study. The expressions of Brn-3α, AR, ERα, components of AKT/m-TOR signaling pathway genes were performed by real-time PCR. The dependence of Brn-3α expression on mRNA levels of steroid hormone receptors and components of AKT/m-TOR signaling pathway in studied cancers were shown. High levels of mRNA of nuclear factor, steroid hormone receptors were found followed by the activation of this signaling pathway in prostate cancer tissue. The reduction of transcription factor Brn-3α was accompanied with tumor invasive growth with increasing rates of AR, ER and 4E-BP1 mRNA. Thyroid cancer development happened in a case of a Brn-3α and steroid hormone receptors decrease. The activation of AKT/m-TOR signaling pathway was established in the metastatic renal cancers, accompanied with the increase of ER mRNA. But there was no correlation between the steroid receptor and Brn-3α. One-direction changes of Brn-3α were observed in the development of prostate and thyroid cancer due to its effect on the steroid hormone receptors and the activation of AKT/m-TOR signaling pathway components. The influence of this factor on the development of the kidney cancer was mediated through m-TOR activity modifications, the key enzyme of oncogenesis.

LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

PubMed Central

Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

2009-01-01

Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

PubMed Central

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-01-01

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

PubMed

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Rubber Elongation Factor (REF), a Major Allergen Component in Hevea brasiliensis Latex Has Amyloid Properties

PubMed Central

Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Cullin, Christophe; Deffieux, Alain; Peruch, Frédéric

2012-01-01

REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom. PMID:23133547

Rubber elongation factor (REF), a major allergen component in Hevea brasiliensis latex has amyloid properties.

PubMed

Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Cullin, Christophe; Deffieux, Alain; Peruch, Frédéric

2012-01-01

REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.

Hydrogen sulfide upregulated mRNA expressions of sodium bicarbonate cotransporter1, trefoil factor1 and trefoil factor2 in gastric mucosa in rats.

PubMed

Cheraghi, Parisa; Mard, Seyyed Ali; Nagi, Tahereh

2016-01-01

Hydrogen sulfide (H 2 S) has been shown to protect the gastric mucosa through several protective mechanisms but till now its effect on mRNA expression of sodium bicarbonate cotransporter 1 (NBC1), trefoil factor1 (TFF1) and trefoil factor2 (TFF2) was not investigated. This study was aimed to evaluate the effect of H 2 S on mRNA expression of NBC1, TFF1 and TFF2 in rat gastric mucosa in response to gastric distention. Thirty two rats were randomly assigned into four equal groups. They were control (C), distention (D), propargylglycine (PAG)-, and NaHS-treated groups. To evaluate the effect of exogenous and endogenous H 2 S on gene expression of NBC1, TFF1 and TFF2, two groups of rats were received H 2 S donor, intra-peritoneal NaHS (80 µg Kg -1 ), and PAG (50 mg kg -1 ), accompanied to stimulate the gastric acid secretion, respectively. Under general anesthesia and laparotomy, a catheter was inserted into the stomach through duodenum for instillation of isotonic saline for gastric distention. Ninety min after beginning the experiment, animals were sacrificed and the gastric mucosa was collected to determine total acid content of gastric effluents and to quantify the mRNA expression of studied genes by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that A) gastric distention increased the level of mRNA expressions of NBC1, TFF1 and TFF2; B) these levels in NaHS-treated rats were significantly higher than those in Distention group; and C) PAG decreased the expression levels of NBC1 and TFF1. The Findings showed H 2 S upregulated gene expression of NBC1, TFF1 and TFF2 in gastric mucosa.

Rapid regulation of brain-derived neurotrophic factor mRNA within eye-specific circuits during ocular dominance column formation.

PubMed

Lein, E S; Shatz, C J

2000-02-15

The neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a candidate retrograde signaling molecule for geniculocortical axons during the formation of ocular dominance columns. Here we examined whether neuronal activity can regulate BDNF mRNA in eye-specific circuits in the developing cat visual system. Dark-rearing throughout the critical period for ocular dominance column formation decreases levels of BDNF mRNA within primary visual cortex, whereas short-term (2 d) binocular blockade of retinal activity with tetrodotoxin (TTX) downregulates BDNF mRNA within the lateral geniculate nucleus (LGN) and visual cortical areas. Brief (6 hr to 2 d) monocular TTX blockade during the critical period and also in adulthood causes downregulation in appropriate eye-specific laminae in the LGN and ocular dominance columns within primary visual cortex. Monocular TTX blockade at postnatal day 23 also downregulates BDNF mRNA in a periodic fashion, consistent with recent observations that ocular dominance columns can be detected at these early ages by physiological methods. In contrast, 10 d monocular TTX during the critical period does not cause a lasting decrease in BDNF mRNA expression in columns pertaining to the treated eye, consistent with the nearly complete shift in physiological response properties of cortical neurons in favor of the unmanipulated eye known to result from long-term monocular deprivation. These observations demonstrate that BDNF mRNA levels can provide an accurate "molecular readout" of the activity levels of cortical neurons and are consistent with a highly local action of BDNF in strengthening and maintaining active synapses during ocular dominance column formation.

Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs

PubMed Central

Sudou, Norihiro; Garcés-Vásconez, Andrés; López-Latorre, María A.; Taira, Masanori

2016-01-01

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin–insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs. PMID:27140624

REF Onida Approval

EPA Pesticide Factsheets

This update August 9, 2016 letter from EPA approves, with modifications, the petition from Ring-neck Energy & Feed, LLC, REF Onida facility, with modifications, regarding non-grandfathered ethanol produced through a dry mill process

Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

PubMed

Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

2016-03-01

A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action. Copyright © 2016 Elsevier B.V. All rights reserved.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

PubMed Central

Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K

2014-01-01

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed. PMID:25033834

Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol.

PubMed

Gabriel, Kara I; Glavas, Maria M; Ellis, Linda; Weinberg, Joanne

2005-06-09

Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90-110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.

Aiding and Abetting Cancer: mRNA export and the nuclear pore

PubMed Central

Culjkovic-Kraljacic, Biljana; Borden, Katherine L.B

2013-01-01

mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter export of specific transcripts encoding proteins involved in proliferation, survival and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance. PMID:23582887

[Transcription factors NF-kB, HIF-1, HIF-2, growth factor VEGF, VEGFR2 and carboanhydrase IX mRNA and protein level in the development of kidney cancer metastasis].

PubMed

Spirina, L V; Usynin, Y A; Yurmazov, Z A; Slonimskaya, E M; Kolegova, E S; Kondakova, I V

2017-01-01

Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.

Differential restriction patterns of mRNA decay factor AUF1 during picornavirus infections.

PubMed

Cathcart, Andrea L; Semler, Bert L

2014-07-01

During infection by picornaviruses, the cellular environment is modified to favour virus replication. This includes the modification of specific host proteins, including the recently discovered viral proteinase cleavage of mRNA decay factor AU-rich binding factor 1 (AUF1). This cellular RNA-binding protein was shown previously to act as a restriction factor during poliovirus, rhinovirus and coxsackievirus infection. During infection by these viruses, AUF1 relocalizes to the cytoplasm and is cleaved by the viral 3C/3CD proteinase. In this study, we demonstrated that replication of encephalomyocarditis virus (EMCV), a picornavirus belonging to the genus Cardiovirus, is AUF1 independent. During EMCV infection, AUF1 relocalized to the cytoplasm; however, unlike what is seen during enterovirus infections, AUF1 was not cleaved to detectable levels, even at late times after infection. This suggests that AUF1 does not act broadly as an inhibitor of picornavirus infections but may instead act as a selective restriction factor targeting members of the genus Enterovirus. © 2014 The Authors.

Nucleotide sequence and regulatory studies of VGF, a nervous system-specific mRNA that is rapidly and relatively selectively induced by nerve growth factor.

PubMed

Salton, S R

1991-09-01

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.

The Antagonistic Effect of Selenium on Cadmium-Induced Damage and mRNA Levels of Selenoprotein Genes and Inflammatory Factors in Chicken Kidney Tissue.

PubMed

Wang, Xinyue; Bao, Rongkun; Fu, Jing

2018-02-01

Selenium (Se) is a necessary trace mineral in the diet of humans and animals. Cadmium (Cd) is a toxic heavy metal that can damage animal organs, especially the kidneys. Antagonistic interactions between Se and Cd have been reported in previous studies. However, little is known about the effects of Se against Cd toxicity and on the mRNA levels of 25 selenoprotein genes and inflammatory factors in chicken kidneys. In the current study, we fed chickens with a Se-treated, Cd-treated, or Se/Cd treated diet for 90 days. We then analyzed the mRNA expression of inflammatory factors (including prostaglandin E synthase (PTGES), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and 25 selenoprotein genes (Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, Dio3, SPS2, Sepp1, SelPb, Sep15, Selh, Seli, Selm, Selo, Sels, Sepx1, Selu, Selk, Selw, Seln, Selt). The results demonstrated that Cd exposure increased the Cd content in the chicken kidneys, renal tubular epithelial cells underwent denaturation and necrosis, and the tubules became narrow or disappeared. However, Se supplementation reduced the Cd content in chicken kidneys and induced normal development of renal tubular epithelial cells. In addition, we also observed that Se alleviated the Cd-induced increase in the mRNA levels of inflammatory factors and ameliorated the Cd-induced downtrend in the mRNA levels of 25 selenoprotein genes in chicken kidneys.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

PubMed Central

Aitken, Colin Echeverría; Beznosková, Petra; Vlčkova, Vladislava; Chiu, Wen-Ling; Zhou, Fujun; Valášek, Leoš Shivaya; Hinnebusch, Alan G; Lorsch, Jon R

2016-01-01

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001 PMID:27782884

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Clinical significance of serum circulating insulin-like growth factor-1 (IGF-1) mRNA in hepatocellular carcinoma.

PubMed

Karabulut, S; Duranyıldız, D; Tas, F; Gezer, U; Akyüz, F; Serilmez, M; Ozgür, E; Yasasever, C T; Vatansever, S; Aykan, N F

2014-03-01

The principal aim of our study was to investigate the usefulness of serum protein and circulating mRNA of insulin-like growth factor-1 (IGF-1) as a diagnostic and prognostic tool in hepatocellular carcinoma (HCC). Fifty-four HCC patients and age- and sex-matched 20 healthy controls were enrolled into this study. Pretreatment serum IGF-1 and IGF-1 mRNA were determined by the solid-phase sandwich ELISA and quantitative RT-PCR method, respectively. The median age at diagnosis was 60 years, range 36-77 years; where majority of group were male (n = 48, 88.8%). All patients had cirrhotic history. Forty-six percent (n = 25) of patients had Child-Pugh score A, 30% (n = 16) had score B or C. All of the patients were treated with local therapies and none of them received sorafenib. The baseline serum IGF-1 mRNA levels were significantly higher in HCC patients than in the control group (p = 0.04), whereas no significant difference was observed for IGF-1 protein levels between the two group (p = 0.18). Patients with history of HBV infection, who were not treated, and who received multiple palliative treatment for HCC had higher serum IGF-1 mRNA levels (p = 0.03, 0.03, and 0.05, respectively). Poor performance status (p < 0.001), viral etiology of cirrhosis (p = 0.03), larger tumor size (p = 0.01), lower serum hemoglobin levels (p = 0.03), and not be treated for HCC (p = 0.001) related to worse survival. However, neither serum IGF-1 nor serum IGF-1 mRNA had significantly adverse effect on survival (p = 0.53 and 0.42, respectively).

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration.

PubMed

Cespedes, Isabel C; de Oliveira, Amanda R; da Silva, Joelcimar M; da Silva, André V; Sita, Luciane V; Bittencourt, Jackson C

2010-12-01

Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1-3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger-Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam. Copyright © 2010 Elsevier Inc. All rights reserved.

An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress.

PubMed

Huch, Susanne; Nissan, Tracy

2017-03-14

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.

APE/Ref-1 is increased in nuclear fractions of human thyroid hyperfunctioning nodules.

PubMed

Russo, D; Celano, M; Bulotta, S; Bruno, R; Arturi, F; Giannasio, P; Filetti, S; Damante, G; Tell, G

2002-08-30

Apurinic/apyrimidinic endonuclease APE/Ref-1 is a multifunctional protein provided with DNA repair, transcription-factor regulation and anti-apoptotic activities. We have previously reported that, in thyroid cells, TSH regulates both the synthesis and nuclear translocation of APE/Ref-1. We have also shown that nuclear levels of this protein are reduced both in thyroid carcinoma tissues and cell lines. In the present study, APE/Ref-1 expression and cellular localization were analysed by Western blot in hyperfunctioning thyroid nodules from patients with toxic adenoma and/or toxic multinodular goiter. The total content of APE/Ref-1 protein was increased in the majority of the hyperfunctioning tissues with respect to normal adjacent tissue. There was also an increase in the nuclear levels of APE/Ref-1, suggesting enhanced cytoplasm-to-nucleus translocation of the protein in addition to its increased rate of synthesis. These results demonstrate that the phenomenon of nuclear translocation of APE/Ref-1 hypothesized on the basis of cell culture experiments does actually occur in vivo. Together with previous observations in thyroid carcinomas and tumoral cell lines, our findings suggest a two-stage model of APE/Ref-1 behaviour during malignant thyrocyte transformation: an early stage characterized by simple hyperplasia and upregulation of APE/Ref-1 in the nuclear compartment of the cell and a later stage in which nuclear levels of the protein drop to below-normal levels as the cell becomes progressively undifferentiated.

The association of the placental Hypoxia-inducible factor1-α polymorphisms and HIF1-α mRNA expression with preeclampsia.

PubMed

Harati-Sadegh, Mahdiyeh; Kohan, Leila; Teimoori, Batool; Mehrabani, Mehrnaz; Salimi, Saeedeh

2018-07-01

Evidence has confirmed that placental/fetal hypoxia plays a key role in both endothelial cell dysfunction and PE pathogenesis. The aim of the present study was to investigate whether maternal/placental hypoxia-inducible factor1-α (HIF1-α) C1772T (rs11549465) and/or G1790A (rs11549467) polymorphisms and HIF1-α mRNA expression are associated with PE development. The blood samples of 203 PE and 202 control women and the placenta of 86 PE and 84 control women were collected after delivery. The HIF1-α polymorphisms were genotyped using PCR- RFLP method. The mRNA expression levels were measured by Quantitative Real -Time PCR. The present study found no association between maternal HIF1-α rs11549465 and rs11549467 and placental rs11549467 polymorphisms and PE. However, the placental rs11549465 polymorphism was associated with PE in the dominant model. The CT/GG combined genotypes and TG haplotype of placental rs11549465 and rs11549467 polymorphisms were associated with higher risk of PE. The HIF1-α mRNA expression was 3-fold higher in the PE women. The rs11549465 TT genotype was associated with higher HIF1-α mRNA expression in PE women and in total population and rs11549467 GA genotype was associated with higher mRNA expression in total population. The relative mRNA expression of HIF1-α gene was higher in presence of CC/GA, TT/GG and TT/GA combined genotypes. This study found an association between placental but not maternal HIF1-α rs11549465 polymorphism and PE in the dominant model. The HIF1-α mRNA expression was higher in the placenta of PE women and was associated with rs11549465 and rs11549467 polymorphisms. Copyright © 2018. Published by Elsevier Ltd.

Absolute Measurements of Macrophage Migration Inhibitory Factor and Interleukin-1-β mRNA Levels Accurately Predict Treatment Response in Depressed Patients.

PubMed

Cattaneo, Annamaria; Ferrari, Clarissa; Uher, Rudolf; Bocchio-Chiavetto, Luisella; Riva, Marco Andrea; Pariante, Carmine M

2016-10-01

Increased levels of inflammation have been associated with a poorer response to antidepressants in several clinical samples, but these findings have had been limited by low reproducibility of biomarker assays across laboratories, difficulty in predicting response probability on an individual basis, and unclear molecular mechanisms. Here we measured absolute mRNA values (a reliable quantitation of number of molecules) of Macrophage Migration Inhibitory Factor and interleukin-1β in a previously published sample from a randomized controlled trial comparing escitalopram vs nortriptyline (GENDEP) as well as in an independent, naturalistic replication sample. We then used linear discriminant analysis to calculate mRNA values cutoffs that best discriminated between responders and nonresponders after 12 weeks of antidepressants. As Macrophage Migration Inhibitory Factor and interleukin-1β might be involved in different pathways, we constructed a protein-protein interaction network by the Search Tool for the Retrieval of Interacting Genes/Proteins. We identified cutoff values for the absolute mRNA measures that accurately predicted response probability on an individual basis, with positive predictive values and specificity for nonresponders of 100% in both samples (negative predictive value=82% to 85%, sensitivity=52% to 61%). Using network analysis, we identified different clusters of targets for these 2 cytokines, with Macrophage Migration Inhibitory Factor interacting predominantly with pathways involved in neurogenesis, neuroplasticity, and cell proliferation, and interleukin-1β interacting predominantly with pathways involved in the inflammasome complex, oxidative stress, and neurodegeneration. We believe that these data provide a clinically suitable approach to the personalization of antidepressant therapy: patients who have absolute mRNA values above the suggested cutoffs could be directed toward earlier access to more assertive antidepressant strategies

Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats.

PubMed

Iwasa, Takeshi; Matsuzaki, Toshiya; Yano, Kiyohito; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Yiliyasi, Maira; Kuwahara, Akira; Irahara, Minoru

2016-11-01

The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours' fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats' serum leptin levels were decreased by 24h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy

Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm

PubMed Central

TATSUNO, TAKANORI; NAKAMURA, YUKA; MA, SHAOFU; TOMOSUGI, NAOHISA; ISHIGAKI, YASUHITO

2016-01-01

Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immune-purified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2-binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti-Upf2 and anti-RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells. PMID:27221324

Effect of electroacupuncture on brain-derived neurotrophic factor mRNA expression in mouse hippocampus following cerebral ischemia-reperfusion injury.

PubMed

Zhao, Jianxin; Xu, Huazhou; Tian, Yuanxiang; Hu, Manxiang; Xiao, Hongling

2013-04-01

This work aims to observe the effects of electroacupuncture on brain-derived neurotrophic factor (BDNF) mRNA expression in mouse hippocampus following cerebral ischemia-reperfusion injury. The models of mouse cerebral ischemia-reperfusion injury were established. A total of 96 healthy mice were randomly assigned into 4 groups, namely, the sham surgery, model, model + electroacupuncture, and mode + hydergine groups. Mice in the model + electroacupuncture group were treated through electroacupuncture at the Shenshu (BL 23), Geshu (BL 17), and Baihui (GV 20) acupoints. Mice in the model+hydergine group were intragastrically administered with hydergine (0.77 mg/kg(-1) x day(-1)). The levels of BDNF mRNA expressions in the hippocampus were ana lyzed through a semi-quantitative reverse transcription-polymerase chain reaction assay on days 1 and 7 after the surgeries. BDNF mRNA expressions in the mouse hippocampus of the model group on days 1 and 7 after the surgery were higher than those of the sham surgery group (both P < 0.01). On days 1 and 7 of the electroacupuncture treatment, BDNF mRNA expression in the mouse hippocampus of the model + electroacupuncture group was significantly elevated compared with the model group (both P < 0.01) or the model + hydergine group (both P < 0.01). On days 1 and 7 of the hydergine treatment, BDNF mRNA expression in the mouse hippocampus of the model + hydergine group tended to increase compared with the model group; however, statistical significance was not achieved (both P > 0.05). Electroacupuncture treatment enhances endogenous BDNF expression, which may improve the survival environment for intracerebral neurons and inhibit the apoptosis of hippocampal cells.

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

PubMed

Salton, S R; Fischberg, D J; Dong, K W

1991-05-01

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Staufen-mediated mRNA decay.

PubMed

Park, Eonyoung; Maquat, Lynne E

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

Staufen-mediated mRNA decay

PubMed Central

Park, Eonyoung; Maquat, Lynne E.

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

PubMed

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-10-15

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. © 2016. Published by The Company of Biologists Ltd.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

PubMed Central

Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

2016-01-01

ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

Expression of CD30 mRNA, CD30L mRNA and a variant form of CD30 mRNA in restimulated peripheral blood mononuclear cells (PBMC) of patients with helminthic infections resembling a Th2 disease

PubMed Central

Kilwinski, J; Berger, T; Mpalaskas, J; Reuter, S; Flick, W; Kern, P

1999-01-01

It has been proposed that CD30, a member of the tumour necrosis factor (TNF) receptor superfamily, is preferentially up-regulated on Th2-type human T cells. In order to investigate a correlation between infection with Echinococcus multilocularis and CD30 expression, we analysed regulation of CD30 mRNA, a variant form of CD30 mRNA (CD30v) and CD30 ligand (CD30L) mRNA expression on PBMC from patients with alveolar echinococcosis (AE) using reverse transcriptase-polymerase chain reaction (RT-PCR). In PBMC of patients with AE as well as healthy donors, spontaneous expression of CD30L mRNA and the CD30v mRNA could be detected. However, the intact form of CD30 mRNA could be detected neither in freshly isolated PBMC of patients nor in PBMC of healthy individuals. Expression of CD30L mRNA and the variant form of CD30 mRNA was frequently detected at individual time points during 72 h of culture of PBMC stimulated with crude Echinococcus antigen. In contrast to CD30v or CD30L mRNA expression, induction of CD30 mRNA expression was detected only in three out of six (50%) healthy donors and in 10 out of 21 (48%) patients with alveolar echinococcosis after 72 h of incubation. As a control, mitogenic stimulation of PBMC of both healthy individuals and infected patients led to expression of intact CD30 mRNA within 24 h of culture. These data demonstrate the different expression of two different forms of CD30 mRNA in PBMC of human individuals. The specific induction of CD30 expression is correlated only in rare cases with the clinical status of patients with AE, indicating the lack of a general induction of CD30 mRNA in this Th2-type-dominated helminthic disease. The data provide further evidence that the CD30 receptor is not an exclusive marker for a Th2-type response. PMID:9933429

Multiple Transcript Properties Related to Translation Affect mRNA Degradation Rates in Saccharomyces cerevisiae

PubMed Central

Neymotin, Benjamin; Ettorre, Victoria; Gresham, David

2016-01-01

Degradation of mRNA contributes to variation in transcript abundance. Studies of individual mRNAs have shown that both cis and trans factors affect mRNA degradation rates. However, the factors underlying transcriptome-wide variation in mRNA degradation rates are poorly understood. We investigated the contribution of different transcript properties to transcriptome-wide degradation rate variation in the budding yeast, Saccharomyces cerevisiae, using multiple regression analysis. We find that multiple transcript properties are significantly associated with variation in mRNA degradation rates, and that a model incorporating these properties explains ∼50% of the genome-wide variance. Predictors of mRNA degradation rates include transcript length, ribosome density, biased codon usage, and GC content of the third position in codons. To experimentally validate these factors, we studied individual transcripts expressed from identical promoters. We find that decreasing ribosome density by mutating the first translational start site of a transcript increases its degradation rate. Using coding sequence variants of green fluorescent protein (GFP) that differ only at synonymous sites, we show that increased GC content of the third position of codons results in decreased rates of mRNA degradation. Thus, in steady-state conditions, a large fraction of genome-wide variation in mRNA degradation rates is determined by inherent properties of transcripts, many of which are related to translation, rather than specific regulatory mechanisms. PMID:27633789

Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Eric; Jakinovich, Paul; Bae, Aekyung

Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1}more » knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is

[Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

PubMed

Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

2012-01-01

The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

PubMed

Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

2015-06-01

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

PubMed Central

Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

2016-01-01

The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

[Effects of Guilin Watermelon Frost on the mRNA expressions of basic fibroblast growth factor in patients with uterine cervical columnar ectopy].

PubMed

Qiu-Yan, Jiang; Jin-Ling, Song; Hai-Xia, Mo

2012-01-01

To study the molecular biological effects of Guilin Watermelon Frost (GWF) on the mRNA expressions of basic fibroblast growth factor (bFGF) in patients with uterine uterine cervical columnar ectopy. One hundred and sixty patients with uterine cervical columnar ectopy were assigned to two groups by the random digit table. Patients in the treatment group were treated with local spray of GWF, while those in the control group were local applied with bFGF-collagen sponge. The mRNA expressions of bFGF of the uterine tissue were detected in the two groups before and after treatment using RT-PCR. Before treatment the mRNA expression of bFGF in the uterine cervical columnar ectopy was 0.55 +/- 0.10 in the treatment group and 0.58 +/- 0.13 in the control group, without insignificant difference (P > 0.05). After treatment it significantly increased in the two groups, being 0.82 +/- 0.17 and 0.78 +/- 0.15 respectively, showing statistical difference from before treatment (P < 0.01). But no statistical difference existed between the two groups after treatment (P > 0.05). GWF showed enhancement on the mRNA expressions of bFGF in patients with uterine cervical columnar ectopy.

Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

PubMed

Fraser, Christopher S

2015-07-01

The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Modification of tissue-factor mRNA and protein response to thrombin and interleukin 1 by high glucose in cultured human endothelial cells.

PubMed

Boeri, D; Almus, F E; Maiello, M; Cagliero, E; Rao, L V; Lorenzi, M

1989-02-01

Because diabetic vascular disease is accompanied by a state of hypercoagulability, manifested by increased thrombin activity and foci of intravascular coagulation, we investigated whether a specific procoagulant property of the endothelium--production and surface expression of tissue factor--is modified by elevated glucose concentrations. In unperturbed human vascular endothelial cells, tissue factor mRNA and expression of the functional protein were undetectable and were not induced by 10-12 days of exposure to 30 mM glucose. In thrombin-stimulated cultures, tissue-factor expression was related inversely to cellular density, with confluent cultures producing (per 10(5) cells) half the amount of tissue factor measured in sparse cultures. Cells exposed to high glucose and studied when cell number and thymidine incorporation were identical to control cells manifested increased tissue-factor mRNA level and functional protein production in response to thrombin (P = .002). This effect was not attributable to hypertonicity and was not observed after short exposure to high glucose. In contrast, the tissue-factor response to interleukin 1, a modulator of endothelial function in the context of host defense, was decreased in cells cultured in high glucose (P = .04). These findings indicate that exposure to high glucose can alter tissue-factor gene expression in perturbed vascular endothelium. The reciprocal effects of high glucose on the tissue-factor response to thrombin and interleukin 1 points to different pathways of tissue-factor stimulation by the two agents and suggests functional consequences pertinent to the increased thrombin activity and compromised host-defense mechanisms observed in diabetes.

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

PubMed

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

A disintegrin and metalloproteinase 17 mRNA and protein expression in esophageal squamous cell carcinoma, as well as its clinicopathological factors and prognosis

PubMed Central

LIU, HONG-BIN; YANG, QI-CHANG; SHEN, YI; ZHU, YAN; ZHANG, XIAO-JUAN; CHEN, HAO

2015-01-01

The aim of the present study was to explore a disintegrin and metalloproteinase 17 (ADAM17) mRNA and protein expression in esophageal squamous cell carcinoma and its association with clinicopathological factors and prognosis. Through semi-quantitative reverse transcription polymerase chain reaction, the ADAM17 mRNA expression in 50 cases of esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were detected. Using streptavidin peroxidase conjugated immunohistochemistry, ADAM17 protein levels were detected in 80 cases of esophageal squamous cell carcinoma and corresponding normal esophageal mucosa. A log rank test and the Cox proportional hazards model were used for the esophageal cancer survival analysis. ADAM17 mRNA expression levels in esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were 0.937±0.241 and 0.225±0.077, respectively (P<0.01). ADAM17 mRNA expression in esophageal squamous cell carcinoma was correlated with lymph node metastasis (P<0.01) and tumor, node and metastasis (TNM) staging (P<0.05), however, it was not correlated with gender, age or histological grade (P>0.05). ADAM17 protein expression rates in esophageal squamous cell carcinoma and corresponding normal esophageal mucosa were 66.25 and 6.25% respectively, a difference that was statistically significant (P<0.01). In addition, ADAM17 protein expression in esophageal squamous cells was correlated with lymph node metastasis and TNM stage (P<0.05), while it was not correlated with gender, age or histological grade (P>0.05). ADAM17 protein expression and epidermal growth factor receptor (EGFR) protein expression were positively correlated (P<0.01). Lymph node metastasis, TNM stage, ADAM17 and EGFR protein expression may be used as independent prognostic indicators of esophageal squamous cell carcinoma (all P<0.05). ADAM17 mRNA and protein were highly expressed in esophageal squamous cell carcinoma; they have important roles in invasion and

Biomaterials for mRNA Delivery

PubMed Central

Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

2015-01-01

Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

Interconnections between mRNA degradation and RDR-dependent siRNA production in mRNA turnover in plants.

PubMed

Tsuzuki, Masayuki; Motomura, Kazuki; Kumakura, Naoyoshi; Takeda, Atsushi

2017-03-01

Accumulation of an mRNA species is determined by the balance between the synthesis and the degradation of the mRNA. Individual mRNA molecules are selectively and actively degraded through RNA degradation pathways, which include 5'-3' mRNA degradation pathway, 3'-5' mRNA degradation pathway, and RNA-dependent RNA polymerase-mediated mRNA degradation pathway. Recent studies have revealed that these RNA degradation pathways compete with each other in mRNA turnover in plants and that plants have a hidden layer of non-coding small-interfering RNA production from a set of mRNAs. In this review, we summarize the current information about plant mRNA degradation pathways in mRNA turnover and discuss the potential roles of a novel class of the endogenous siRNAs derived from plant mRNAs.

Changes in mRNA levels for brain-derived neurotrophic factor after wheel running in rats selectively bred for high- and low-aerobic capacity

PubMed Central

Groves-Chapman, Jessica L.; Murray, Patrick S.; Stevens, Kristin L.; Monroe, Derek; Koch, Lauren G.; Britton, Steven L.; Holmes, Philip V.

2012-01-01

We evaluated levels of exercise-induced brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) within the hippocampal formation in rats selectively bred for 1) high intrinsic (i.e., untrained) aerobic capacity (High Capacity Runners, HCR), 2) low intrinsic aerobic capacity (Low Capacity Runners, LCR), and 3) unselected Sprague-Dawley (SD) rats with or without free access to running wheels for three weeks. The specific aim of the study was to determine whether a dose-response relationship exists between cumulative running distance and levels of BDNF mRNA. No additional treatments or behavioral manipulations were used. HCR, LCR, and SD rats were grouped by strain and randomly assigned to sedentary or activity (voluntary access to activity wheel) conditions. Animals were killed after 21 days of exposure to the assigned conditions. Daily running distances (mean ± standard deviation meters/d) during week three were: HCR (4726 ± 3220), SD (2293 ± 3461), LCR (672 ± 323). Regardless of strain, levels of BDNF mRNA in CA1 were elevated in wheel runners compared to sedentary rats and this difference persisted after adjustment for age (p=0.040). BDNF mRNA was not affected by intrinsic aerobic capacity and was not related to total running distance. The results support that BDNF mRNA expression is increased by unlimited access to activity wheel running for 3 weeks but is not dependent upon accumulated running distance. PMID:22024546

Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

PubMed

Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

2018-01-05

The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficacy of Omega Fatty Acid Supplementation on mRNA Expression Level of Tumor Necrosis Factor Alpha in Patients with Gastric Adenocarcinoma.

PubMed

Hosseinzadeh, Asghar; Ardebili, Seyed Mojtaba Mohaddes

2016-09-01

Tumor necrosis factor alpha (TNF-α), a multifunctional cytokine, is involved in apoptosis, cell proliferation, cell survival, and inflammation. It plays a dual role in cancer development and progression. It has been revealed that polyunsaturated fatty acids (PUFAs) modulate the production and activity of TNF family cytokines. The objective of the present study was to evaluate the effect of PUFAs on messenger RNA expression levels of TNF-α in patients with gastric adenocarcinoma. Thirty-four chemotherapy-naive patients diagnosed with gastric adenocarcinoma were randomly divided into two groups. The first group (17 individuals) received cisplatin without supplements and the second group (17 individuals) received cisplatin plus orally administered PUFA supplements for 3 weeks, based on treatment strategies. The gastric biopsy samples were obtained from all participants before and after treatment, and TNF-α mRNA expression levels were evaluated by quantitative real-time PCR procedure. Our findings revealed that TNF-α mRNA expression is downregulated in group II, after receiving cisplatin and omega fatty acid supplement for 3 weeks. However, this difference is not statistically significant (p > 0.05). TNF-α mRNA expression did not show significant alteration in group I, after receiving cisplatin alone. Taken together, we concluded that omega fatty acids reduce TNF-α expression at the mRNA level in patients with gastric adenocarcinoma. These data suggest that TNF-α may act as a potential target for the therapy of human gastric adenocarcinoma.

Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

PubMed

Danner, S; Lohse, M J

1997-07-16

Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

Assessing Impact Submissions for REF 2014: An Evaluation

ERIC Educational Resources Information Center

Manville, Catriona; Guthrie, Susan; Henham, Marie-Louise; Garrod, Bryn; Sousa, Sonia; Kirtley, Anne; Castle-Clarke, Sophie; Ling, Tom

2015-01-01

The Research Excellence Framework (REF) is a new system for assessing the quality of research in UK higher education institutions (HEIs). For the first time, part of the assessment included the wider impact of research. RAND Europe was commissioned to evaluate the assessment process of the impact element of REF submissions, and to explore the…

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

PubMed Central

Akool, El-Sayed; Kleinert, Hartmut; Hamada, Farid M. A.; Abdelwahab, Mohamed H.; Förstermann, Ulrich; Pfeilschifter, Josef; Eberhardt, Wolfgang

2003-01-01

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA. PMID:12832476

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

PubMed

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10

PubMed Central

Pyeon, Dohun; Splitter, Gary A.

1999-01-01

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10

Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

PubMed

Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Zhendre, Vanessa; Henry, Sarah; Thévenot, Julie; Dufourc, Erick J; Alves, Isabel D; Peruch, Frédéric

2014-01-01

The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles. © 2013.

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

PubMed

Han, L Q; Zhou, Z; Ma, Y; Batistel, F; Osorio, J S; Loor, J J

2018-04-18

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14

Molar loss and powder diet leads to memory deficit and modifies the mRNA expression of brain-derived neurotrophic factor in the hippocampus of adult mice.

PubMed

Takeda, Yosuke; Oue, Hiroshi; Okada, Shinsuke; Kawano, Akira; Koretake, Katsunori; Michikawa, Makoto; Akagawa, Yasumasa; Tsuga, Kazuhiro

2016-12-05

It is known that tooth loss is known to be a risk factor for Alzheimer's disease and soft diet feeding induces memory impairment. Recent studies have shown that brain-derived neurotrophic factor (BDNF) is associated with tooth loss or soft diet in young animal model, and that BDNF expression is decreased in patients with Alzheimer's disease. However, single or combined effect of tooth loss and/or soft diet on brain function has not fully understood. Here we examined the effect of molar loss and powder diet on memory ability and the expression of BDNF mRNA in the hippocampus of adult C57BL/6J mice. Twenty eight-weeks-old C57BL/6J mice were divided into intact molar group and extracted molar group. They were randomly divided into the I/S group (Intact upper molar teeth/Solid diet feeding), the E/S group (Extracted upper molar teeth/Solid diet feeding), the I/P group (Intact upper molar teeth/Powder diet feeding), and the E/P group (Extracted upper molar teeth/Powder diet feeding). The observation periods were 4 and 16-week. To analyze the memory ability, the step-through passive avoidance test was conducted. BDNF-related mRNA in the hippocampus was analyzed by real-time polymerase chain reaction (RT-PCR). At 4 weeks later, we performed memory test and isolated brains to analyze. There were no differences in memory function and BDNF mRNA level between these four groups. However, at 16 weeks later, E/S and E/P group showed memory impairment, and decreased level of BDNF mRNA. Whereas, the powder diet had no effect on memory function and BDNF mRNA level even at 16 weeks later. These results suggest that the effect of molar loss and powder diet on memory function and BDNF mRNA levels were different, molar loss may have a greater long-term effect on memory ability than powder diet does.

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

PubMed

Nakashima, Yukiko; Takahashi, Satoru

2014-08-22

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Chitosan nanoparticle carrying small interfering RNA to platelet-derived growth factor B mRNA inhibits proliferation of smooth muscle cells in rabbit injured arteries.

PubMed

Xia, He; Jun, Ji; Wen-Ping, Ling; Yi-Feng, Pan; Xiao-Ling, Chen

2013-10-01

The purpose of this study was to elucidate the transfection of chitosan nanoparticle carrying small interfering RNA against platelet-derived growth factor B (PDGF-B) to inhibit the expression of PDGF-B mRNA and proliferation of smooth muscle cells. A rabbit iliac artery injury model was constructed. A small interfering RNA (siRNA) against PDGF-B mRNA expression vector was constructed and packaged by chitosan nanoparticle to transfect into the vascular smooth muscle cells (vSMCs) of balloon catheter-injured rabbit iliac artery wall, using a therapeutic ultrasound for the gene delivery. The experiment was divided into two groups: experimental group, denudation and nano-PDGF-B siRNA treated, and only single denudation as control. Effects of the siRNA on the expressions of proliferating cell nuclear antigen (PCNA) and PDGF-B mRNA by vSMCs and the proliferation of vSMCs were observed with the methods of routine pathological, immunohistochemical staining, in situ hybridization and morphometry. The nano siRNA against PDGF-B was successfully transfected. The nano siRNA significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal vSMCs. The local intimal thickness and area were also reduced remarkably. In conclusion, transfection of chitosan nanoparticle carrying siRNA against PDGF-B mRNA could inhibit proliferation of vSMCs in the rabbit iliac artery injury model. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Cytokine dysregulation in AIDS: in vivo overexpression of mRNA of tumor necrosis factor-alpha and its correlation with that of the inflammatory cytokine GRO.

PubMed

Dezube, B J; Pardee, A B; Beckett, L A; Ahlers, C M; Ecto, L; Allen-Ryan, J; Anisowicz, A; Sager, R; Crumpacker, C S

1992-01-01

The human immunodeficiency virus establishes an intimate interaction with the immune system. The virus can use cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (Il-1), to regulate its own expression by modifying the normal immunoregulatory network. We demonstrate that mRNA of the cytokine TNF-alpha from peripheral blood mononuclear cells is overexpressed in virtually all patients with AIDS who do not have active opportunistic infections compared with uninfected volunteers (p < 0.0001). This overexpression correlates with elevated mRNA levels of the recently discovered GRO (p < 0.05), a cytokine involved in the inflammatory response.

Mechanism of Cytoplasmic mRNA Translation

PubMed Central

2015-01-01

Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

The criterion and discriminant validity of the Referential Thinking (REF) scale.

PubMed

Startup, Mike; Sakrouge, Rebecca; Mason, Oliver J

2010-03-01

The Referential Thinking (REF) scale was designed to be a comprehensive self-report measure of both simple and guilty ideas of reference in the general population. One aim of the present study was to test the proposed interpretations of REF scores by comparing REF scores with ratings of delusions among psychotic patients. A 2nd aim was to test whether REF scores are better predicted by the severity of patients' delusions of reference (DoRs) than by the severity of their auditory verbal hallucinations (AVHs), thus supporting the scores' ability to discriminate between proneness to the 2 different symptoms. The REF scale was completed by 56 healthy controls and 53 acutely psychotic patients. The severity of the patients' DoRs and AVHs were assessed in structured clinical interviews. REF scores differed significantly not only between the patients and controls but also between patients with versus without DoRs. REF scores correlated significantly with the severity of the patients' DoRs but not their AVHs. The interpretation of REF scores as a measure of proneness to simple and guilty ideas of reference was supported. PsycINFO Database Record (c) 2010 APA, all rights reserved.

In utero and lactational exposure to low-dose genistein-vinclozolin mixture affects the development and growth factor mRNA expression of the submandibular salivary gland in immature female rats.

PubMed

Kouidhi, Wided; Desmetz, Catherine; Nahdi, Afef; Bergès, Raymond; Cravedi, Jean-Pierre; Auger, Jacques; El May, Michèle; Canivenc-Lavier, Marie Chantal

2012-06-01

It has been suggested that hormonally controlled submandibular salivary gland (SSG) development and secretions may be affected by endocrine disruptor compounds. We investigated the effects of oral gestation-lactation exposure to 1 mg/kg body weight daily dose of the estrogenic soy-isoflavone genistein and/or the anti-androgenic food contaminant vinclozolin in female rats. The SSGs of female offspring were collected at postnatal day 35 to study gland morphogenesis and mRNA expression of sex-hormone receptors and endocrine growth factors as sex-dependent biomarkers. Because of high expression in neonatal SSG, mRNA expression of transforming growth factor α was also studied. Exposure to genistein, vinclozolin, or a genistein+vinclozolin mixture resulted in significantly lower numbers of striated ducts linked to an increase in their area and lower acinar proliferation (Ki-67-positive nuclei). Exposure to the mixture had the highest significant effects, which were particularly associated with repression of epidermal growth factor, nerve growth factor, and transforming growth factor α expression. In conclusion, early exposure to low doses of genistein and vinclozolin can affect glandular structure and endocrine gene mRNA expression in prepubertal SSG in female rats, and the effects are potentialized by the genistein+vinclozolin mixture. Our study provides the first evidence that SSG are targeted by both estrogenic and anti-androgenic disrupting compounds and are more sensitive to mixtures.

Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

PubMed Central

Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

2017-01-01

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics

PubMed Central

Velazquez, J R; Lacy, P; Moqbel, R

2000-01-01

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released

Discovering mutated driver genes through a robust and sparse co-regularized matrix factorization framework with prior information from mRNA expression patterns and interaction network.

PubMed

Xi, Jianing; Wang, Minghui; Li, Ao

2018-06-05

Discovery of mutated driver genes is one of the primary objective for studying tumorigenesis. To discover some relatively low frequently mutated driver genes from somatic mutation data, many existing methods incorporate interaction network as prior information. However, the prior information of mRNA expression patterns are not exploited by these existing network-based methods, which is also proven to be highly informative of cancer progressions. To incorporate prior information from both interaction network and mRNA expressions, we propose a robust and sparse co-regularized nonnegative matrix factorization to discover driver genes from mutation data. Furthermore, our framework also conducts Frobenius norm regularization to overcome overfitting issue. Sparsity-inducing penalty is employed to obtain sparse scores in gene representations, of which the top scored genes are selected as driver candidates. Evaluation experiments by known benchmarking genes indicate that the performance of our method benefits from the two type of prior information. Our method also outperforms the existing network-based methods, and detect some driver genes that are not predicted by the competing methods. In summary, our proposed method can improve the performance of driver gene discovery by effectively incorporating prior information from interaction network and mRNA expression patterns into a robust and sparse co-regularized matrix factorization framework.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

PubMed

Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

2017-10-01

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones.

PubMed

Tong, Zheng; Wang, Dan; Sun, Yong; Yang, Qian; Meng, Xueru; Wang, Limin; Feng, Weiqiang; Li, Ling; Wurtele, Eve Syrkin; Wang, Xuchu

2017-05-02

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF 138,175,258 and SRPP 117,204,243 , were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF 258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF 175 or REF 258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

Laser-induced thermotherapy (LITT) elevates mRNA expression of connective tissue growth factor (CTGF) associated with reduced tumor growth of liver metastases compared to hepatic resection.

PubMed

Isbert, Christoph; Ritz, Jörg-Peter; Roggan, André; Schuppan, Detlef; Ajubi, Navid; Buhr, Heinz Johannes; Hohenberger, Werner; Germer, Christoph-Thomas

2007-01-01

Proliferation and synthesis of hepatocellular tissue after tissue damage are promoted by specific growth factors such as hepatic tissue growth factor (HGF) and connective growth factor (CTGF). Laser-induced thermotherapy (LITT) for the treatment of liver metastases is deemed to be a parenchyma-saving procedure compared to hepatic resection. The aim of this study was to compare the impact of LITT and hepatic resection on intrahepatic residual tumor tissue and expression levels of mRNA HGF/CTGF within liver and tumor tissue. Two independent adenocarcinomas (CC531) were implanted into 75 WAG rats, one in the right (untreated tumor) and one in the left liver lobe (treated tumor). The left lobe tumor was treated either by LITT or partial hepatectomy. The control tumor was submitted to in-situ hybridization of HGF and CTGF 24-96 hours and 14 days after intervention. Volumes of the untreated tumors prior to intervention were 38+/-8 mm(3) in group I (laser), 39 +/- 7 mm(3) in group II (resection), and 42 +/- 12 mm(3) in group III (control) and did not differ significantly (P > 0.05). Fourteen days after the intervention the mean tumor+/-SEM volume of untreated tumor in group I (laser) [223 +/- 36] was smaller than in group II (resection) [1233.28 +/- 181.52; P < 0.001], and in group III (control) [978.92 +/- 87.57; P < 0.003]. Forty-eight hours after the intervention intrahepatic mRNA expression level of HGF in group II (resection) was almost twofold higher than in group I (laser) [7.2 +/- 1.0 c/mf vs. 3.9 +/- 0.4 c/mf; P<0.01]. Fourteen days after the intervention intrahepatic mRNA expression level of CTGF in group I (laser) was higher than in group II (resection) [13.89 +/- 0.77 c/mf vs. 9.09 +/- 0.78 c/mf; P < 0.003]. LITT leads to a decrease of residual tumor growth in comparison to hepatic resection. Accelerated tumor growth after hepatic resection is associated with higher mRNA level of HGF and reduced tumor growth after LITT with higher mRNA level of CTGF. The

Expression of hypoxia-inducible factor 1α mRNA in hearts and lungs of broiler chickens with ascites syndrome induced by excess salt in drinking water.

PubMed

Zhang, Jianjun; Feng, Xuejian; Zhao, Lihong; Wang, Wei; Gao, Mingyu; Wu, Boning; Qiao, Jian

2013-08-01

Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed heterodimeric transcription factor that mediates adaptive responses to hypoxia in all nucleated cells of metazoan organisms. Hypoxia-inducible factor 1α is involved in the pathogenesis of pulmonary hypertension in humans and animals, but whether HIF-1α is associated with the development of pulmonary hypertension syndrome (also known as ascites syndrome, AS) in broiler chickens has not been determined. In the present paper we addressed this issue by measuring the expression of HIF-1α mRNA in hearts and lungs of broiler chickens with AS induced by excess salt in drinking water. We conducted 2 experiments. The first experiment was used to observe the effects of excess salt on AS incidence. The results indicated that total incidence (20%) of AS in excess salt group (receiving 0.3% NaCl in drinking water) was much higher compared with the control group (receiving tap water) over a 43-d time course (P < 0.05). In the second experiment, we determined mean pulmonary arterial pressure (mPAP), ascites heart index (AHI), and expression of HIF-1α mRNA in lungs and hearts of broiler chickens after the excess salt treatment. Our results showed that excess salt induced pulmonary hypertension (indicated by higher mPAP) and right ventricular hypertrophy (greater ascites heart index) in broiler chickens. Meanwhile, the expression levels of HIF-1α mRNA in lungs and hearts were significantly increased at different time points in the excess salt group compared with the control group. Linear correlation analysis showed that the expression of HIF-1α mRNA in lungs was significantly positively correlated with mPAP (correlation coefficient = 0.79, P < 0.001), demonstrating that expression of HIF-1α mRNA was gradually increased in the excess salt group with the increase of pulmonary arterial pressure. In addition, the ascitic chickens showed significantly higher transcriptional levels of HIF-1α in hearts and lungs

mRNA Cancer Vaccines-Messages that Prevail.

PubMed

Grunwitz, Christian; Kranz, Lena M

2017-01-01

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Principles of mRNA transport in yeast.

PubMed

Heym, Roland Gerhard; Niessing, Dierk

2012-06-01

mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas

PubMed Central

Fontanini, G; Boldrini, L; Chinè, S; Pisaturo, F; Basolo, F; Calcinai, A; Lucchi, M; Mussi, A; Angeletti, C A; Bevilacqua, G

1999-01-01

The vascular endothelial growth factor (VEGF) has been shown to be strictly related to vascular permeability and endothelial cell growth under physiological and pathological conditions. In tumour development and progression, VEGF plays a pivotal role in the development of the tumoral vascular network, and useful information in the progression of human cancer can be obtained by analysing the vascular endothelial growth factor expression of the tumours. In this study, we investigated the vascular endothelial growth factor transcript expression in non-small-cell lung carcinomas to evaluate the significance of this factor in a group of cancers in which the vascular pattern has been shown to significantly affect progression. Surgical samples of 42 patients with NSCLC were studied using reverse transcription polymerase chain reaction (PCR) analysis and in situ hybridization. Thirty-three out of 42 cases (78.6%) showed VEGF transcript expression predominantly as transcripts for the secretory forms of VEGF (isoforms 121 and 165). In situ hybridization, performed on 24 out of 42 samples, showed that the VEGF transcript expression was in several cases present in the cytoplasm both of neoplastic and normal cells, even if the VEGF mRNA was less expressed in the corresponding non-tumoral part. The VEGF 121 expression was associated with hilar and/or mediastinal nodal involvement (P = 0.02), and, taken together, the VEGF isoforms were shown to significantly influence overall (P = 0.02) and disease-free survival (P = 0.03). As a regulator of tumour angiogenesis, VEGF may represent a useful indicator of progression and poor prognosis in non-small-cell lung carcinomas. © 1999 Cancer Research Campaign PMID:9888482

Postage for the messenger: Designating routes for Nuclear mRNA Export

PubMed Central

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

PubMed Central

Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

Macrophage colony-stimulating factor accelerates wound healing and upregulates TGF-beta1 mRNA levels through tissue macrophages.

PubMed

Wu, L; Yu, Y L; Galiano, R D; Roth, S I; Mustoe, T A

1997-10-01

Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 microg/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF-beta1 mRNA level in M-CSF-treated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N = 21, P < 0.01) and 50% (P < 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF-beta1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N = 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF-beta1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF-beta. The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is adversely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level. Copyright 1997 Academic Press.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers.

PubMed

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-02-17

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.

Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors.

PubMed

Tanner, Julie-Anne; Prasad, Bhagwat; Claw, Katrina G; Stapleton, Patricia; Chaudhry, Amarjit; Schuetz, Erin G; Thummel, Kenneth E; Tyndale, Rachel F

2017-01-01

Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine. Copyright © 2016 by The American Society for Pharmacology and

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

PubMed

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Hyoung; Lim, Mi-Hyun; Youn, Dong-Ye

We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). Amore » super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.« less

Platelet-derived growth factor A mRNA in platelets is associated with the degree of hepatic fibrosis in chronic hepatitis C.

PubMed

Tanikawa, Aline Aki; Grotto, Rejane Maria Tommasini; Silva, Giovanni Faria; Ferrasi, Adriana Camargo; Sarnighausen, Valéria Cristina Rodrigues; Pardini, Maria Inês de Moura Campos

2017-01-01

Transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor (PDGF) are the main cytokines related to hepatic fibrogenesis. RNA isolated from the platelets and hepatic tissue of 43 HCV carriers was used for quantitative polymerase chain reaction to determine TGFB1, PDGFA, and PDGFB RNA expression. The mRNA expression of PDGFA in platelets was significantly lower in the group with advanced fibrosis than in the group with early-stage fibrosis. TGFB1 was more frequently expressed in platelets than in hepatic tissue, which was different from PDGFB. A pathway mediated by overexpression of TGFB1 via PDGFA in megakaryocytes could be involved in the development of fibrosis.

The Criterion and Discriminant Validity of the Referential Thinking (REF) Scale

ERIC Educational Resources Information Center

Startup, Mike; Sakrouge, Rebecca; Mason, Oliver J.

2010-01-01

The Referential Thinking (REF) scale was designed to be a comprehensive self-report measure of both simple and guilty ideas of reference in the general population. One aim of the present study was to test the proposed interpretations of REF scores by comparing REF scores with ratings of delusions among psychotic patients. A 2nd aim was to test…

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii.

PubMed

Serpeloni, Mariana; Jiménez-Ruiz, Elena; Vidal, Newton Medeiros; Kroeber, Constanze; Andenmatten, Nicole; Lemgruber, Leandro; Mörking, Patricia; Pall, Gurman S; Meissner, Markus; Ávila, Andréa R

2016-11-01

Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans. © 2016 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii

PubMed Central

Serpeloni, Mariana; Jiménez‐Ruiz, Elena; Vidal, Newton Medeiros; Kroeber, Constanze; Andenmatten, Nicole; Lemgruber, Leandro; Mörking, Patricia; Pall, Gurman S.

2016-01-01

Summary Nucleo‐cytoplasmic RNA export is an essential post‐transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans. PMID:27542978

Vibrational force alters mRNA expression in osteoblasts

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

1997-01-01

Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

TSHB mRNA is linked to cholesterol metabolism in adipose tissue.

PubMed

Moreno-Navarrete, José María; Moreno, María; Ortega, Francisco; Xifra, Gemma; Hong, Shangyu; Asara, John M; Serrano, José C E; Jové, Mariona; Pissios, Pavlos; Blüher, Matthias; Ricart, Wifredo; Portero-Otin, Manuel; Fernández-Real, José Manuel

2017-10-01

Subclinical hypothyroidism is known to be associated with increased serum cholesterol. Since thyroid-stimulating hormone (TSH) exerts an inductor effect on cholesterol biosynthesis, we aimed to investigate the relationship between TSH mRNA and cholesterol metabolism in human adipose tissue (AT). Cross-sectionally, AT TSH-β ( TSHB ) mRNA was evaluated in 4 independent cohorts in association with serum total and LDL cholesterol, and AT lipidomics. Longitudinally, the effects of statins and of diet and exercise on AT TSHB mRNA were also examined. The bidirectional relationship between cholesterol and TSHB were studied in isolated human adipocytes. TSHB mRNA was consistently detected in AT from euthyroid subjects, and positively associated with serum total- and LDL-cholesterol, and with AT-specific cholesterol metabolism-associated lipids [arachidonoyl cholesteryl ester, C8-dihydroceramide, N -stearoyl-d-sphingosine, and GlcCer(18:0, 24:1)]. Reduction of cholesterol with statins and with diet and exercise interventions led to decreased TSHB mRNA in human AT, whereas excess cholesterol up-regulated TSHB mRNA in human adipocytes. In addition, recombinant human TSH α/β administration resulted in increased HMGCR mRNA levels in human adipocytes. In mice, subcutaneous AT Tshb expression levels correlated directly with circulating cholesterol levels. In summary, current results provide novel evidence of TSHB as a paracrine factor that is modulated in parallel with cholesterol metabolism in human AT.-Moreno-Navarrete, J. M., Moreno, M., Ortega, F., Xifra, G., Hong, S., Asara, J. M., Serrano, J. C. E., Jové, M., Pissios, P., Blüher, M., Ricart, W., Portero-Otin, M., Fernández-Real, J. M. TSHB mRNA is linked to cholesterol metabolism in adipose tissue. © FASEB.

Control of sigma virus multiplication by the ref(2)P gene of Drosophila melanogaster: an in vivo study of the PB1 domain of Ref(2)P.

PubMed

Carré-Mlouka, A; Gaumer, S; Gay, P; Petitjean, A M; Coulondre, C; Dru, P; Bras, F; Dezélée, S; Contamine, D

2007-05-01

Ref(2)P has been described as one of the Drosophila proteins that interacts with the sigma virus cycle. We generated alleles to identify critical residues involved in the restrictive (inhibiting viral multiplication) or permissive (allowing viral multiplication) character of Ref(2)P. We demonstrate that permissive alleles increase the ability of the sigma virus to infect Drosophila when compared to null alleles and we confirm that restrictive alleles decrease this capacity. Moreover, we have created alleles unfunctional in viral cycling while functional for Ref(2)P fly functions. This type of allele had never been observed before and shows that fly- and virus-related activities of Ref(2)P are separable. The viral status of Ref(2)P variants is determined by the amino-terminal PB1 domain polymorphism. In addition, an isolated PB1 domain mimics virus-related functions even if it is similar to a loss of function toward fly-related activities. The evolutionary tree of the Ref(2)P PB1 domain that we could build on the basis of the natural allele sequences is in agreement with an evolution of PB1 domain due to successive transient selection waves.

Abundance of mRNA of growth hormone receptor and insulin-like growth factors-1 and -2 in duodenal and colonic biopsies of dogs with chronic enteropathies*.

PubMed

Spichiger, A C; Allenspach, K; Ontsouka, E; Gaschen, F; Morel, C; Blum, J W; Sauter, S N

2005-12-01

Repair processes of the inflamed intestine are very important for dissolution of chronic enteropathies (CE). Therefore, we examined the mRNA abundance of growth hormone receptor (GHR), insulin-like growth factors (IGF)-1 and -2 in duodenal and colonic biopsies of dogs with CE such as food-responsive diarrhoea (FRD) and inflammatory bowel disease (IBD) before and after treatment as compared with each other and healthy dogs. A clinical score (Canine IBD Activity Index = CIBDAI) was applied to judge the severity of CE. Biopsies of duodenum and colon from client-owned dogs with CE were sampled before (FRD(bef), n = 5; IBD(bef), n = 5) and after treatment (FRD(aft), n = 5; IBD(aft), n = 5). Intestinal control samples were available from a homogenous control population (n = 15; C). Intestinal samples were homogenized, total RNA was extracted, reverse transcribed and analysed by real-time polymerase chain reaction to measure mRNA levels of GHR, IGF-1 and IGF-2. Results were normalized with glyceraldehyde phosphate dehydrogenase as housekeeping gene. The CIBDAI decreased during the treatment period in FRD and IBD (P < 0.01). In duodenum, GHR mRNA levels were higher in all groups than in C (P < 0.001). Duodenal IGF-1 mRNA levels in FRD(aft) and IBD(aft) tended to be higher than in C (P < 0.1). The IGF-2 mRNA abundance in FRD(aft) was higher than in C (P < 0.05) in duodenum. In colon, mRNA levels of IGF-1 in IBD(aft) were higher than in FRD(aft) (P < 0.05) and levels differed between IBD(aft) and C (P < 0.05). In conclusion, mRNA levels of GHR, IGF-1 and IGF-2 in the gastrointestinal tract were increased during CE when compared with gastrointestinally healthy dogs. The data suggest that GHR, IGF-1 and IGF-2 are involved in gastrointestinal repair processes.

Altered Sex Hormone Concentrations and Gonadal mRNA Expression Levels of Activin Signaling Factors in Hatchling Alligators From a Contaminated Florida Lake

PubMed Central

MOORE, BRANDON C.; KOHNO, SATOMI; COOK, ROBERT W.; ALVERS, ASHLEY L.; HAMLIN, HEATHER J.; WOODRUFF, TERESA K.; GUILLETTE, LOUIS J.

2014-01-01

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin βA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly. PMID:20166196

Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus)

PubMed Central

Shahed, Asha; McMichael, Carling F.; Young, Kelly A.

2017-01-01

This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2(PT day-2), 4(PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptorsα and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation. PMID:26174001

Overcoming Drug Resistant Prostate Cancer with APE1/Ref 1 Blockade

DTIC Science & Technology

2016-10-01

prostate cancer specimens. Genetic knockdown of APE1/Ref-1 disrupts prostate cancer cell growth and survival in cell culture. In addition...inhibition of the redox function selectively of Ref-1 results in cell growth inhibition, with this therapy preferentially inhibiting prostate cancer cell... growth above that in non-cancerous cells. Specific blockade of Ref-1 redox activity in tumors is a novel concept in tumor therapy. If we are successful

Messenger RNA (mRNA) nanoparticle tumour vaccination

NASA Astrophysics Data System (ADS)

Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

2014-06-01

Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

REF4 and RFR1, Subunits of the Transcriptional Coregulatory Complex Mediator, Are Required for Phenylpropanoid Homeostasis in Arabidopsis*

PubMed Central

Bonawitz, Nicholas D.; Soltau, Whitney L.; Blatchley, Michael R.; Powers, Brendan L.; Hurlock, Anna K.; Seals, Leslie A.; Weng, Jing-Ke; Stout, Jake; Chapple, Clint

2012-01-01

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs. PMID:22167189

REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis.

PubMed

Bonawitz, Nicholas D; Soltau, Whitney L; Blatchley, Michael R; Powers, Brendan L; Hurlock, Anna K; Seals, Leslie A; Weng, Jing-Ke; Stout, Jake; Chapple, Clint

2012-02-17

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.

Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation

PubMed Central

Vincent, Heather A.; Ziehr, Benjamin; Moorman, Nathaniel J.

2016-01-01

mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells. PMID:27089357

Subcellular mRNA localisation at a glance

PubMed Central

Parton, Richard M.; Davidson, Alexander; Davis, Ilan; Weil, Timothy T.

2014-01-01

ABSTRACT mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms. PMID:24833669

Chaperone Hsp27 Modulates AUF1 Proteolysis and AU-Rich Element-Mediated mRNA Degradation▿

PubMed Central

Knapinska, Anna M.; Gratacós, Frances M.; Krause, Christopher D.; Hernandez, Kristina; Jensen, Amber G.; Bradley, Jacquelyn J.; Wu, Xiangyue; Pestka, Sidney; Brewer, Gary

2011-01-01

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues—Ser15, Ser78, and Ser82—by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2–Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization. PMID:21245386

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

PubMed Central

2011-01-01

Background One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. Methods The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. Results We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. Conclusions These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development. PMID:21329510

Regulation of cytoplasmic mRNA decay

PubMed Central

Schoenberg, Daniel R.; Maquat, Lynne E.

2012-01-01

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means to modulate gene expression and thereby protein production. Up until recently, studies focused largely on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay, and the ribonucleases that catalyze decay. Now, current studies have begun to elucidate how the decay process is regulated. This review examines our current understanding of how mammalian-cell mRNA decay is controlled by different signaling pathways and lays out a framework for future research. PMID:22392217

Purification of Growth Factor mRNA in Renal Tissues:bFGF-2, FGF-2, TGFα, and EGFR.

PubMed

Mydlo, J H

2001-01-01

Growth factors are polypeptides that induce cell mitogenicity, and thus play an important role in the etiology and progression of tumors (1). Fibroblast growth factors (FGF) constitute a family of structurally related polypeptides of 146 amino acids, which exhibit a wide spectrum of biologic activities, including angiogenesis or the formation of a vascular network. FGFs are mitogenic towards many mesodermal and ectodermal cell types, and can also induce and/or inhibit differentiation of cells (2). These heparin-binding factors are categorized as FGF-1 through FGF-10. Acidic FGF, or FGF-1, is found mostly in brain and other neural tissues. Basic FGF, or FGF- 2, a protein of 18 kDa mw, is one of the most ubiqitous growth factors. It is found in numerous benign and cancerous human and animal tissues, including kidney, prostate, and bladder (3-6). In some cases it has also been demonstrated to have potential as a tumor marker (7-11). One group reported greater recovery of both FGF-2 protein and FGF-2 mRNA from renal-cancer tissue compared to equal amounts of normal renal tissue (5). Furthermore, when purified FGF-2 from renal cell carcinoma (RCC) is added exogenously to other established renal tumorcell lines and endothelial cell lines, it demonstrates significant mitogenic activity (6). Thus, renal tumors may use FGF-2 in an autocrine manner to sustain themselves.

Increase of CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

PubMed

Kunzmann, Steffen; Krempl, Christine; Seidenspinner, Silvia; Glaser, Kirsten; Speer, Christian P; Fehrholz, Markus

2018-04-16

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase of CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Low-level laser therapy (LLLT) attenuates RhoA mRNA expression in the rat bronchi smooth muscle exposed to tumor necrosis factor-alpha.

PubMed

de Lima, Flávia Mafra; Bjordal, Jan M; Albertini, Regiane; Santos, Fábio V; Aimbire, Flavio

2010-09-01

Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Bronchial smooth muscle (BSM) hyperreactivity is associated with increased Ca+2 sensitivity and increased RhoA mRNA expression. In the current study, we investigated if LLLT could reduce BSM contraction force and RhoA mRNA expression in tumor necrosis factor-alpha (TNF-alpha)-induced BSM hyperreactivity. In the study, 112 male Wistar rats were divided randomly into 16 groups, and BSM was harvested and suspended in TNF-alpha baths for 6 and 24 h, respectively. Irradiation with LLLT was performed with a wavelength of 660 nm for 42 s with a dose of 1.3 J/cm2. This LLLT dose was administered once in the 6-h group and twice in the 24-h group. LLLT significantly decreased contraction force in BSM at 6 h (TNF-alpha + LLLT: 11.65+/-1.10 g/100 mg of tissue) (F=3115) and at 24 h (TNF-alpha+ LLLT: 14.15+/-1.1 g/100 mg of tissue) (F=3245, p<0.05) after TNF-alpha, respectively, when compared to vehicle-bathed groups (control). LLLT also significantly decreased the expression of RhoA mRNA in BSM segments at 6 h (1.22+/-0.20) (F=2820, p<0.05) and 24 h (2.13+/-0.20) (F=3324, p<0.05) when compared to BSM segments incubated with TNF-alpha without LLLT irradiation. We conclude that LLLT administered with this protocol, reduces RhoA mRNA expression and BSM contraction force in TNF-alpha-induced BSM hyperreactivity.

Preparing Impact Submissions for REF 2014: An Evaluation. Research Brief

ERIC Educational Resources Information Center

Manville, Catriona; Jones, Molly Morgan; Frearson, Michael; Castle-Clarke, Sophie; Henham, Marie-Louise; Gunashekar, Salil; Grant, Jonathan

2015-01-01

The Research Excellence Framework (REF) 2014 is a nationwide initiative designed to assess the quality of research in UK higher education institutions (HEIs). For the first time, REF 2014 introduced the wider impact of research, alongside the quality of research and the vitality of the research environment, into the assessment of research…

Leptin receptor mRNA in rat brain astrocytes

PubMed Central

Hsuchou, Hung; Pan, Weihong; Barnes, Maria J.; Kastin, Abba J.

2009-01-01

We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in-situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6 h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-α. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response. PMID:19747514

beta-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures.

PubMed

Ye, Ping; Hu, Qichen; Liu, Hedi; Yan, Yun; D'ercole, A Joseph

2010-07-01

By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether beta-catenin, a molecule that is a downstream target of glycogen synthase kinase-3beta (GSK3beta) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases beta-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3beta. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in beta-catenin and cyclin D1 mRNA, while suppression of GSK3beta activity simulated IGF-I actions. Knocking-down beta-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that beta-catenin is an important downstream molecule in the PI3-Akt-GSK3beta pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. Copyright 2010 Wiley-Liss, Inc.

Differential mRNA expression of neuroinflammatory modulators in the spinal cord and thalamus of type 2 diabetic monkeys.

PubMed

Ding, Huiping; Kiguchi, Norikazu; Kishioka, Shiroh; Ma, Tao; Peters, Christopher M; Ko, Mei-Chuan

2018-05-11

Given that diabetes-associated complications are closely associated with neuroinflammation, it is imperative to study potential changes in neuroinflammatory modulators in the central nervous system of diabetic primates. The mRNA levels of pro- and anti-inflammatory cytokines, toll-like receptors (TLRs), growth factors, and cannabinoid receptors were compared in the spinal dorsal horn (SDH) and thalamus of naturally occurring type 2 diabetic monkeys and an age-matched control group using reverse transcription and quantitative real-time polymerase chain reaction. In the SDH of diabetic monkeys, mRNA levels of proinflammatory cytokines (i.e. interleukin [IL]-1β and tumor necrosis factor [TNF] α), TLR1, and TLR2 were increased, whereas mRNA levels of IL-10, an anti-inflammatory cytokine, were decreased. No changes were observed in the mRNA levels of growth factors and cannabinoid receptors. In line with the mRNA data, TNFα immunoreactivity was significantly increased in diabetic monkeys. Moreover, mRNA expression levels of IL-1β, TNFα, TLR1, and TLR2 in the SDH were positively correlated with plasma glucose concentrations in all monkeys. Several ligands and receptors involved in neuroinflammation are simultaneously dysregulated in the spinal cord of diabetic monkeys. This primate disease model will facilitate the design of novel treatment approaches to ameliorate neuroinflammation-driven adverse effects in diabetic patients. © 2018 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Fibroblast growth factor-21 and omentin-1 hepatic mRNA expression and serum levels in morbidly obese women with non-alcoholic fatty liver disease.

PubMed

Waluga, M; Kukla, M; Zorniak, M; Kajor, M; Liszka, L; Dyaczynski, M; Kowalski, G; Zadlo, D; Waluga, E; Olczyk, P; Buldak, R J; Berdowska, A; Hartleb, M

2017-06-01

Fibroblast growth factor-21 (FGF21) and omentin-1 have been recognized as potent antidiabetic agents with potential hepatoprotective activity. The aim of this study was to evaluate hepatic FGF21 and omentin-1 mRNA expression as well as their serum levels as predictive markers of liver injury and insulin resistance in morbidly obese women with non-alcoholic fatty liver disease (NAFLD). This study included 56 severely obese women who underwent intraoperative wedge liver biopsy during the bariatric surgery. Hepatic FGF21 and omentin-1 mRNA were assessed by quantitative real-time PCR, while their serum concentrations were measured with commercially available enzyme-linked immunosorbent assays. The FGF21 serum level was significantly higher in patients with a greater extent of steatosis (grade 2 and 3) compared to those without or with mild steatosis (grade 0 and 1) (P = 0.049). Receiver Operating Characteristic analysis, however, showed poor discriminant power for the FGF21 serum levels in differentiating between more and less extensive steatosis with an AUC = 0.666. There was a tendency towards higher levels of hepatic FGF21 mRNA in patients with lobular inflammation and fibrosis and towards lower levels in the case of hepatocyte ballooning and steatosis. There was a positive mutual correlation between hepatic FGF21 and omentin-1 mRNA levels (r = 0.78; P < 0.001). Fibrosis stage was associated with serum glucose and homeostatic model assessment for insulin resistance (HOMA-IR) (P = 0.03 and P = 0.02, respectively). Serum omentin-1 was not associated with histopathological features. The hepatic omentin-1 mRNA levels showed a tendency to be lower in patients with advanced steatosis and hepatocyte ballooning. In conclusion, our study, which focused on hepatic FGF21 and omentin-1 mRNA expression, confirmed marked expression of both molecules in the liver of morbidly obese patients with NAFLD. More extensive steatosis was associated with evident changes in the serum FGF21

Conceptual Modeling in Systems Biology Fosters Empirical Findings: The mRNA Lifecycle

PubMed Central

Dori, Dov; Choder, Mordechai

2007-01-01

One of the main obstacles to understanding complex biological systems is the extent and rapid evolution of information, way beyond the capacity individuals to manage and comprehend. Current modeling approaches and tools lack adequate capacity to model concurrently structure and behavior of biological systems. Here we propose Object-Process Methodology (OPM), a holistic conceptual modeling paradigm, as a means to model both diagrammatically and textually biological systems formally and intuitively at any desired number of levels of detail. OPM combines objects, e.g., proteins, and processes, e.g., transcription, in a way that is simple and easily comprehensible to researchers and scholars. As a case in point, we modeled the yeast mRNA lifecycle. The mRNA lifecycle involves mRNA synthesis in the nucleus, mRNA transport to the cytoplasm, and its subsequent translation and degradation therein. Recent studies have identified specific cytoplasmic foci, termed processing bodies that contain large complexes of mRNAs and decay factors. Our OPM model of this cellular subsystem, presented here, led to the discovery of a new constituent of these complexes, the translation termination factor eRF3. Association of eRF3 with processing bodies is observed after a long-term starvation period. We suggest that OPM can eventually serve as a comprehensive evolvable model of the entire living cell system. The model would serve as a research and communication platform, highlighting unknown and uncertain aspects that can be addressed empirically and updated consequently while maintaining consistency. PMID:17849002

Self-amplifying mRNA vaccines.

PubMed

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

Growth Cone Localization of the mRNA Encoding the Chromatin Regulator HMGN5 Modulates Neurite Outgrowth

PubMed Central

Moretti, Francesca; Rolando, Chiara; Winker, Moritz; Ivanek, Robert; Rodriguez, Javier; Von Kriegsheim, Alex; Taylor, Verdon; Bustin, Michael

2015-01-01

Neurons exploit local mRNA translation and retrograde transport of transcription factors to regulate gene expression in response to signaling events at distal neuronal ends. Whether epigenetic factors could also be involved in such regulation is not known. We report that the mRNA encoding the high-mobility group N5 (HMGN5) chromatin binding protein localizes to growth cones of both neuron-like cells and of hippocampal neurons, where it has the potential to be translated, and that HMGN5 can be retrogradely transported into the nucleus along neurites. Loss of HMGN5 function induces transcriptional changes and impairs neurite outgrowth, while HMGN5 overexpression induces neurite outgrowth and chromatin decompaction; these effects are dependent on growth cone localization of Hmgn5 mRNA. We suggest that the localization and local translation of transcripts coding for epigenetic factors couple the dynamic neuronal outgrowth process with chromatin regulation in the nucleus. PMID:25825524

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

USGS Publications Warehouse

Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

2000-01-01

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

Vascular endothelial growth factor and platelet-derived growth factor are potential angiogenic and metastatic factors in human breast cancer.

PubMed

Anan, K; Morisaki, T; Katano, M; Ikubo, A; Kitsuki, H; Uchiyama, A; Kuroki, S; Tanaka, M; Torisu, M

1996-03-01

Angiogenesis is a prerequisite for tumor growth and metastasis. Tumor angiogenesis may be mediated by several angiogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha, and basic fibroblast growth factor. Differential mRNA expressions of VEGF, PDGF (A chain), transforming growth factor-alpha and basic fibroblast growth factor in 32 primary invasive breast tumors were examined by reverse transcriptase-polymerase chain reaction. We analyzed relationships between mRNA expressions of these angiogenic factors and the degree of angiogenesis, tumor size, and metastasis. Quantification of angiogenesis was achieved by the immunohistochemical staining of endothelial cells with antibody to CD31. VEGF and PDGF-A mRNAs were expressed more frequently in breast tumors than in nontumor breast tissues, whereas no difference was found in expression frequency of either transforming growth factor-alpha or basic fibroblast growth factor mRNA. Vascular counts in tumors correlated with each expression frequency of VEGF and PDGF-A mRNA. PDGF-A mRNA was expressed more frequently in tumors with lymph node metastasis than in those without metastasis. Expression of VEGF and PDGF mRNAs detected by reverse transcriptase-polymerase chain reaction in breast tumors correlates with tumor-related characteristics of angiogenesis and metastatic potential. Analysis of these mRNAs by reverse transcriptase-polymerase chain reaction may be useful for assessing the biologic behavior of a breast tumor before surgical treatment.

Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver

PubMed Central

Wang, Jingkui; Yeung, Jake; Gobet, Cédric; Sobel, Jonathan; Lück, Sarah; Molina, Nacho; Naef, Felix

2018-01-01

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1−/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver. PMID:29432155

Pulsed low-level infrared laser alters mRNA levels from muscle repair genes dependent on power output in Wistar rats

NASA Astrophysics Data System (ADS)

Trajano, L. A. S. N.; Trajano, E. T. L.; Thomé, A. M. C.; Sergio, L. P. S.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

2017-10-01

Satellite cells are present in skeletal muscle functioning in the repair and regeneration of muscle injury. Activation of these cells depends on the expression of myogenic factor 5 (Myf5), myogenic determination factor 1(MyoD), myogenic regulatory factor 4 (MRF4), myogenin (MyoG), paired box transcription factors 3 (Pax3), and 7 (Pax7). Low-level laser irradiation accelerates the repair of muscle injuries. However, data from the expression of myogenic factors have been controversial. Furthermore, the effects of different laser beam powers on the repair of muscle injuries have been not evaluated. The aim of this study was to evaluate the effects of low-level infrared laser at different powers and in pulsed emission mode on the expression of myogenic regulatory factors and on Pax3 and Pax7 in injured skeletal muscle from Wistar rats. Animals that underwent cryoinjury were divided into three groups: injury, injury laser 25 Mw, and injury laser 75 mW. Low-level infrared laser irradiation (904 nm, 3 J cm-2, 5 kHz) was carried out at 25 and 75 mW. After euthanasia, skeletal muscle samples were withdrawn and the total RNA was extracted for the evaluation of mRNA levels from the MyoD, MyoG, MRF4, Myf5, Pax3, and Pax7 gene. Pax 7 mRNA levels did not alter, but Pax3 mRNA levels increased in the injured and laser-irradiated group at 25 mW. MyoD, MyoG, and MYf5 mRNA levels increased in the injured and laser-irradiated animals at both powers, and MRF4 mRNA levels decreased in the injured and laser-irradiated group at 75 mW. In conclusion, exposure to pulsed low-level infrared laser, by power-dependent effect, could accelerate the muscle repair process altering mRNA levels from paired box transcription factors and myogenic regulatory factors.

mRNA–mRNA duplexes that auto-elicit Staufen1-mediated mRNA decay

PubMed Central

Gong, Chenguang; Tang, Yalan; Maquat, Lynne E.

2013-01-01

We report a new mechanism by which human mRNAs crosstalk: an Alu element in the 3'-untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3' UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3' UTR SBS created via mRNA–mRNA base-pairing, we show that SMD targets both mRNAs in the duplex provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the importance of mRNA–mRNA-triggered SMD to the processes of cell migration and invasion. PMID:24056942

Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles.

PubMed

Azad, Milad; Khaledi, Neda; Hedayati, Mehdi

2016-06-15

Skeletal muscle is a highly elastic tissue which can respond to various functional demands by altering fiber-type composition. Exercise affects muscle fiber phenotype. One of the transcription factors that induce fiber-type transition is forkhead box O1 (FOXO1). Since eccentric contraction considered an essential part of exercise, so we are interested to see the effects of eccentric exercise (acute/chronic) on FOXO1 as an important factor of fiber-type transition in rat skeletal muscles. Twenty-four Sprague-Dawley rats (190-235g) were divided to 3 groups of 8 rats: 1) chronic eccentric exercise (CEE), 2) acute eccentric exercise (AEE), and 3) control (C). The exercise groups underwent downhill running protocol. CEE was running on treadmill in 3 days of week for 9 weeks, that slope and duration gradually managed from -4° to -16° and 15 to 90 min, respectively. AEE group was running with 16 m/min on -16° slope for 3 consecutive days that included 18 sets of 5 min with rest interval of 2 min in between. Soleus and super vastus lateralis (SVL) muscles mRNA were analyzed by real-time RT-PCR. SVL FOXO1 mRNA levels increased by 3.92-fold in the AEE and decreased 0.56-fold in the CEE group and were not significant in soleus muscle. In soleus muscle, myosin heavy chain (MHC) IIa, IIx, and IIb decreased in the AEE group and MHC IIa and IIx decreased in the CEE group. In SVL muscle, MHC I, IIa, and IIx increased in the AEE group and MHC IIa and IIX increased in the CEE group. In summary, both acute and chronic eccentric exercise could lead to change in FOXO1 mRNA only in fast SVL muscle of rat and so could induce fiber-type transition in both muscles regardless of changes in expression of FOXO1. So, oxidative stress can play important role in change of FOXO1. Copyright © 2016 Elsevier B.V. All rights reserved.

mRNA export: threading the needle

PubMed Central

Gaouar, Ouassila; Germain, Hugo

2013-01-01

After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress. PMID:23526740

The involvement of mRNA processing factors TIA-1, TIAR, and PABP-1 during mammalian hibernation.

PubMed

Tessier, Shannon N; Audas, Timothy E; Wu, Cheng-Wei; Lee, Stephen; Storey, Kenneth B

2014-11-01

Mammalian hibernators survive low body temperatures, ischemia-reperfusion, and restricted nutritional resources via global reductions in energy-expensive cellular processes and selective increases in stress pathways. Consequently, studies that analyze hibernation uncover mechanisms which balance metabolism and support survival by enhancing stress tolerance. We hypothesized processing factors that influence messenger ribonucleic acid (mRNA) maturation and translation may play significant roles in hibernation. We characterized the amino acid sequences of three RNA processing proteins (T cell intracellular antigen 1 (TIA-1), TIA1-related (TIAR), and poly(A)-binding proteins (PABP-1)) from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), which all displayed a high degree of sequence identity with other mammals. Alternate Tia-1 and TiaR gene variants were found in the liver with higher expression of isoform b versus a in both cases. The localization of RNA-binding proteins to subnuclear structures was assessed by immunohistochemistry and confirmed by subcellular fractionation; TIA-1 was identified as a major component of subnuclear structures with up to a sevenfold increase in relative protein levels in the nucleus during hibernation. By contrast, there was no significant difference in the relative protein levels of TIARa/TIARb in the nucleus, and a decrease was observed for TIAR isoforms in cytoplasmic fractions of torpid animals. Finally, we used solubility tests to analyze the formation of reversible aggregates that are associated with TIA-1/R function during stress; a shift towards the soluble fraction (TIA-1a, TIA-1b) was observed during hibernation suggesting enhanced protein aggregation was not present during torpor. The present study identifies novel posttranscriptional regulatory mechanisms that may play a role in reducing translational rates and/or mRNA processing under unfavorable environmental conditions.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

PubMed Central

Castells-Roca, Laia; García-Martínez, José; Moreno, Joaquín; Herrero, Enrique; Bellí, Gemma; Pérez-Ortín, José E.

2011-01-01

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins. PMID:21364882

Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

NASA Astrophysics Data System (ADS)

Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

2015-10-01

Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

CELFish ways to modulate mRNA decay

PubMed Central

St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

2013-01-01

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa.

PubMed Central

Lory, S

1986-01-01

A DNA probe from an internal fragment of the exotoxin A structural gene was used to study the effects of selected culture conditions on steady-state levels of exotoxin-specific mRNA in Pseudomonas aeruginosa. Cells grown under conditions of iron deprivation began to synthesize and excrete the exotoxin A polypeptide during the late exponential phase of growth and throughout the stationary phase of growth, concomitant with a sharp increase in exotoxin A mRNA pools in P. aeruginosa cells. The addition of iron to the medium resulted in the failure of these cells to synthesize exotoxin A mRNA, despite significantly enhanced growth. The inhibition of the production of exotoxin A and the accumulation of its mRNA by iron was dose dependent, with a half-maximal inhibitory concentration of FeSO4 of 5 to 10 microM. A blockade of the initiation of transcription by rifampin resulted in the decay of exotoxin A mRNA, with a half-life of approximately 8 to 10 min, depending on the media used for growth. The addition of iron to cells actively engaged in exotoxin A synthesis also resulted in a gradual decrease in the amount of this mRNA in bacteria. However, the rate of decline of mRNA induced by iron was relatively slow (half-life, 90 min), with a considerable lag time between the iron addition and the first detectable effect on mRNA. While iron clearly appears to influence the production of exotoxin A at the transcriptional level, the molecular basis of this effect may involve several interacting factors affecting the initiation of transcription and perhaps mRNA turnover. Images PMID:2430950

RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation.

PubMed

Gill, Tina; Cai, Ti; Aulds, Jason; Wierzbicki, Sara; Schmitt, Mark E

2004-02-01

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.

RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA

PubMed Central

Alspach, Elise; Stewart, Sheila A.

2016-01-01

Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014). PMID:27453911

Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

PubMed

Johansson, Marcus J O

2017-01-01

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

Polysome Fractionation to Analyze mRNA Distribution Profiles.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2017-02-05

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al. , 2014a and 2014b; Abdelmohsen et al. , 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al. , 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

PubMed

Ihmann, Thomas; Liu, Jian; Schwabe, Wolfgang; Häusler, Peter; Behnke, Detlev; Bruch, Hans-Peter; Broll, Rainer; Windhövel, Ute; Duchrow, Michael

2004-12-01

The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

PubMed Central

Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

PubMed Central

Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

2014-01-01

SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

PubMed

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

[The expressions of HSP 70 mRNA and c-fos mRNA in the skeletal muscle and cardiac muscle of rabbits by electrocuted].

PubMed

Wang, Ye; Liu, Min; Cheng, Wei-bo; He, Gui-qiong; Li, Fan; Liao, Zhi-gang

2008-08-01

To study the changes of HSP 70 mRNA and c-fos mRNA expression and to find a method to differentiate antemortem from postmortem electrocution. Fifteen New Zealand rabbits were randomly divided into three groups, the antemortem electrocution group, the postmortem electrocution group, and the control group. Each group consists of five rabbits. The levels of HSP 70 mRNA and c-fos mRNA in skeletal muscle and cardiac muscle were examined with quantitative fluorescent RT-PCR. The levels of HSP 70 mRNA and c-fos mRNA in the antemortem electrocution group increased significantly (P<0.05), compared with that of the postmortem electrocution group. The changes of HSP 70 mRNA and c-fos mRNA expression in skeletal muscle and cardiac muscle can be used as an indicator to distinguish antemortem from postmortem electrocution.

Impact in the REF: Issues and Obstacles

ERIC Educational Resources Information Center

Watermeyer, Richard

2016-01-01

This article focuses on "impact" as a new condition of research assessment for UK academics. It explores a history of resistance to an "impact agenda" and how impact as a component of the Research Excellence Framework (REF)--a system of performance based funding--is viewed by academics as an infringement to a scholarly way of…

Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

PubMed

Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

2017-07-01

N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Preparing Impact Submissions for REF 2014: An Evaluation. Findings and Observations

ERIC Educational Resources Information Center

Manville, Catriona; Jones, Molly Morgan; Frearson, Michael; Castle-Clarke, Sophie; Henham, Marie-Louise; Gunashekar, Salil; Grant, Jonathan

2015-01-01

The Research Excellence Framework (REF) 2014 is a nationwide initiative designed to assess the quality of research in UK higher education institutions (HEIs). For the first time, REF 2014 introduced the wider impact of research, alongside the quality of research and the vitality of the research environment, into the assessment of research…

Preparing Impact Submissions for REF 2014: An Evaluation. Approach and Evidence

ERIC Educational Resources Information Center

Manville, Catriona; Jones, Molly Morgan; Henham, Marie-Louise; Castle-Clarke, Sophie; Frearson, Michael; Gunashekar, Salil; Grant, Jonathan

2015-01-01

The Research Excellence Framework (REF) 2014 is a nationwide initiative designed to assess the quality of research in UK higher education institutions (HEIs). For the first time, REF 2014 introduced the wider impact of research, alongside the quality of research and the vitality of the research environment, into the assessment of research…

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.

PubMed Central

Pelletier, J; Kaplan, G; Racaniello, V R; Sonenberg, N

1988-01-01

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction. Images PMID:2836606

Update on RefSeq microbial genomes resources.

PubMed

Tatusova, Tatiana; Ciufo, Stacy; Federhen, Scott; Fedorov, Boris; McVeigh, Richard; O'Neill, Kathleen; Tolstoy, Igor; Zaslavsky, Leonid

2015-01-01

NCBI RefSeq genome collection http://www.ncbi.nlm.nih.gov/genome represents all three major domains of life: Eukarya, Bacteria and Archaea as well as Viruses. Prokaryotic genome sequences are the most rapidly growing part of the collection. During the year of 2014 more than 10,000 microbial genome assemblies have been publicly released bringing the total number of prokaryotic genomes close to 30,000. We continue to improve the quality and usability of the microbial genome resources by providing easy access to the data and the results of the pre-computed analysis, and improving analysis and visualization tools. A number of improvements have been incorporated into the Prokaryotic Genome Annotation Pipeline. Several new features have been added to RefSeq prokaryotic genomes data processing pipeline including the calculation of genome groups (clades) and the optimization of protein clusters generation using pan-genome approach. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

PubMed

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

HER-2 gene amplification, HER-2 and epidermal growth factor receptor mRNA and protein expression, and lapatinib efficacy in women with metastatic breast cancer.

PubMed

Press, Michael F; Finn, Richard S; Cameron, David; Di Leo, Angelo; Geyer, Charles E; Villalobos, Ivonne E; Santiago, Angela; Guzman, Roberta; Gasparyan, Armen; Ma, Yanling; Danenberg, Kathy; Martin, Anne Marie; Williams, Lisa; Oliva, Cristina; Stein, Steven; Gagnon, Robert; Arbushites, Michael; Koehler, Maria T

2008-12-01

Biomarkers from two randomized phase III trials were analyzed to optimize selection of patients for lapatinib therapy. In available breast cancer tissue from EGF30001 (paclitaxel +/- lapatinib in HER-2-negative/unknown metastatic breast cancer, n = 579) and EGF100151 (capecitabine +/- lapatinib in HER-2-positive metastatic breast cancer, n = 399), HER-2 gene amplification by fluorescence in situ hybridization (FISH), HER-2 mRNA by reverse transcription-PCR (RT-PCR), HER-2 protein expression by HercepTest immunohistochemistry (IHC), epidermal growth factor receptor (EGFR) mRNA level by RT-PCR, and EGFR protein by IHC were analyzed and compared with clinical outcome. HER-2 was determined by FISH in an academic reference/research laboratory and in a large, high-volume commercial reference laboratory. The HER-2 gene was amplified in 47% (344 of 733) and IHC was 3+ in 35% (279 of 798), with significant correlation (P < 0.01) between FISH and IHC. Positive EGFR immunostaining (IHC 1+, 2+, or 3+) in 28% (213 of 761) correlated with EGFR mRNA levels by RT-PCR (r = 0.59; P < 0.01). HER-2 gene amplification/overexpression was associated with improved clinical outcomes (progression-free survival; P < 0.001) in both trials. A significant improvement in outcome was seen in FISH-positive and IHC 0, 1+, or 2+ patients. HER-2 mRNA expression correlated with HER-2 FISH (r = 0.83) and IHC status (r = 0.72; n = 138). No correlation was found between EGFR expression (IHC or mRNA) and responsiveness to lapatinib regardless of HER-2 status. Although a significant correlation with lapatinib responsiveness was observed among "HER-2-negative" breast cancer patients in the large, high-volume commercial reference laboratory, this was not confirmed in the academic reference/research laboratory. Women with HER-2-positive metastatic breast cancer benefit from lapatinib, whereas women with HER-2-negative metastatic breast cancer derive no incremental benefit from lapatinib.

Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish.

PubMed

Bouleau, Aurélien; Desvignes, Thomas; Traverso, Juan Martin; Nguyen, Thaovi; Chesnel, Franck; Fauvel, Christian; Bobe, Julien

2014-08-01

The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates. © 2014 by the Society for the Study of Reproduction, Inc.

The ACI-REF Program: Empowering Prospective Computational Researchers

NASA Astrophysics Data System (ADS)

Cuma, M.; Cardoen, W.; Collier, G.; Freeman, R. M., Jr.; Kitzmiller, A.; Michael, L.; Nomura, K. I.; Orendt, A.; Tanner, L.

2014-12-01

The ACI-REF program, Advanced Cyberinfrastructure - Research and Education Facilitation, represents a consortium of academic institutions seeking to further advance the capabilities of their respective campus research communities through an extension of the personal connections and educational activities that underlie the unique and often specialized cyberinfrastructure at each institution. This consortium currently includes Clemson University, Harvard University, University of Hawai'i, University of Southern California, University of Utah, and University of Wisconsin. Working together in a coordinated effort, the consortium is dedicated to the adoption of models and strategies which leverage the expertise and experience of its members with a goal of maximizing the impact of each institution's investment in research computing. The ACI-REFs (facilitators) are tasked with making connections and building bridges between the local campus researchers and the many different providers of campus, commercial, and national computing resources. Through these bridges, ACI-REFs assist researchers from all disciplines in understanding their computing and data needs and in mapping these needs to existing capabilities or providing assistance with development of these capabilities. From the Earth sciences perspective, we will give examples of how this assistance improved methods and workflows in geophysics, geography and atmospheric sciences. We anticipate that this effort will expand the number of researchers who become self-sufficient users of advanced computing resources, allowing them to focus on making research discoveries in a more timely and efficient manner.

Alternative polyadenylation of mRNA precursors

PubMed Central

Tian, Bin; Manley, James L.

2017-01-01

Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

Sensitivity of mRNA Translation

PubMed Central

Poker, Gilad; Margaliot, Michael; Tuller, Tamir

2015-01-01

Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

Monitoring autophagic flux using Ref(2)P, the Drosophila p62 ortholog.

PubMed

DeVorkin, Lindsay; Gorski, Sharon M

2014-09-02

Human p62, also known as Sequestome-1 (SQSTM1), is a multifunctional scaffold protein that contains many domains, including a Phox/Bem1P (PB1) multimerization domain, an ubiquitin-associated (UBA) domain, and a light chain 3 (LC3) recognition sequence. p62 binds ubiquitinated proteins and targets them for degradation by the proteasome. In addition, p62 directly binds LC3; this may serve as a mechanism to deliver ubiquitinated proteins for degradation by autophagy. During this process, p62 itself is degraded. The inhibition of autophagy leads to the accumulation of p62, indicating that it can be used as a marker of autophagic flux. Ref(2)P (refractory to sigma P), the Drosophila ortholog of p62, is also required for the formation of ubiquitinated protein aggregates. Ref(2)P contains a putative LC3-interacting region, and genetic inhibition of autophagy in Drosophila leads to the accumulation of Ref(2)P protein levels. Thus, like p62, Ref(2)P may serve as a marker of autophagic flux. Here we provide two procedures to examine Ref(2)P protein levels in Drosophila ovaries. © 2014 Cold Spring Harbor Laboratory Press.

Multiple cis-acting signals, some weak by necessity, collectively direct robust transport of oskar mRNA to the oocyte.

PubMed

Ryu, Young Hee; Kenny, Andrew; Gim, Youme; Snee, Mark; Macdonald, Paul M

2017-09-15

Localization of mRNAs can involve multiple steps, each with its own cis -acting localization signals and transport factors. How is the transition between different steps orchestrated? We show that the initial step in localization of Drosophila oskar mRNA - transport from nurse cells to the oocyte - relies on multiple cis -acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of oskar mRNA localization. We propose that the oskar transport signals are weak by necessity; their weakness facilitates transfer of the oskar mRNA from the oocyte transport machinery to the machinery for posterior localization. © 2017. Published by The Company of Biologists Ltd.

TNF-α messenger ribonucleic acid (mRNA) in patients with nonalcoholic steatohepatitis.

PubMed

Alaaeddine, Nada; Sidaoui, Joseph; Hilal, George; Serhal, Reem; Abedelrahman, Abir; Khoury, Salem

2012-01-01

tumor necrosis factor (TNF)-α plays a significant role in the pathogenesis of nonalcoholic steatohepatitis (NASH). A few studies have confirmed high TNF-α plasma protein levels in patients with NASH compared to healthy volunteers. We herein aimed to revisit these findings using other molecular techniques. a cross-sectional evaluation of patients newly diagnosed with NASH. A quantitative assay for the measurement of TNF-α messenger ribonucleic acid (mRNA) was performed for NASH patients and controls using real-time reverse transcription polymerase chain reaction (RT-PCR). in 39 patients with NASH (mean age 38.6 ± 9.4 years, range 28-60 years; 79% males), the mean TNF-α mRNA level was significantly higher than that found for controls (137.6 ± 102.3 ng/mL versus 83.5 ± 43.8 ng/mL, respectively; P = 0.012). A TNF-α mRNA cut-off of 100 ng/mL predicted NASH most optimally (AUC 0.685 ± 0.066, P = 0.01; with 66.7% sensitivity and 74.1% specificity). Serum TNF-α and soluble TNF-α receptor II (sTNFRII) levels were significantly higher in patients compared to controls using ELISA. high TNF-α mRNA levels, determined by RT-PCR, characterize patients with NASH.

Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

PubMed

Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

1998-12-01

Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

Learning-induced expression of meningeal ependymin mRNA and demonstration of ependymin in neurons and glial cells.

PubMed

Rother, S; Schmidt, R; Brysch, W; Schlingensiepen, K H

1995-10-01

The turnover of a CNS-specific cell adhesion glycoprotein, ependymin, has earlier been found to increase during periods of neuronal plasticity. Here, ependymin mRNA expression was analyzed by semiquantitative in situ hybridization in goldfish. Learning of an active avoidance response resulted in a significant increase in ependymin mRNA expression 20 min to 4 h after acquisition of the task. In contrast, yoked control animals that were exposed to the same numbers of conditioned and unconditioned stimuli in a random, unpaired manner exhibited a strong down-regulation of ependymin mRNA. Hybridization signals were also increased by injection of anti-ependymin antiserum into brain ventricles. Ependymin mRNA was exclusively localized to reticular-shaped fibroblasts of the inner endomeningeal cell layer. Immunoelectron microscopic investigation, however, revealed ependymin also in distinct neuronal and glial cell populations in which no ependymin mRNA had been detected. Uptake of meningeal protein factors into glial and neuronal cells may therefore be of functional importance for plastic adaptations of the CNS.

Characterization of rat calcitonin mRNA.

PubMed Central

Amara, S G; David, D N; Rosenfeld, M G; Roos, B A; Evans, R M

1980-01-01

A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA. Images PMID:6933496

Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice.

PubMed

Narita, M; Shimamura, M; Imai, S; Kubota, C; Yajima, Y; Takagi, T; Shiokawa, M; Inoue, T; Suzuki, M; Suzuki, T

2008-03-18

The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2

Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

PubMed

Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

2014-05-01

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

PubMed Central

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

PubMed

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

PubMed

Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

PubMed Central

Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M.; Brandl, Holger; Schwich, Oliver D.; Steiner, Michaela C.; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M.

2016-01-01

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1–7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1–7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3′ ends. PMID:26944680

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

PubMed Central

Lackford, Brad; Yao, Chengguo; Charles, Georgette M; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

2014-01-01

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification. PMID:24596251

iRefWeb: interactive analysis of consolidated protein interaction data and their supporting evidence

PubMed Central

Turner, Brian; Razick, Sabry; Turinsky, Andrei L.; Vlasblom, James; Crowdy, Edgard K.; Cho, Emerson; Morrison, Kyle; Wodak, Shoshana J.

2010-01-01

We present iRefWeb, a web interface to protein interaction data consolidated from 10 public databases: BIND, BioGRID, CORUM, DIP, IntAct, HPRD, MINT, MPact, MPPI and OPHID. iRefWeb enables users to examine aggregated interactions for a protein of interest, and presents various statistical summaries of the data across databases, such as the number of organism-specific interactions, proteins and cited publications. Through links to source databases and supporting evidence, researchers may gauge the reliability of an interaction using simple criteria, such as the detection methods, the scale of the study (high- or low-throughput) or the number of cited publications. Furthermore, iRefWeb compares the information extracted from the same publication by different databases, and offers means to follow-up possible inconsistencies. We provide an overview of the consolidated protein–protein interaction landscape and show how it can be automatically cropped to aid the generation of meaningful organism-specific interactomes. iRefWeb can be accessed at: http://wodaklab.org/iRefWeb. Database URL: http://wodaklab.org/iRefWeb/ PMID:20940177

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

PubMed

Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

PubMed Central

Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

2015-01-01

The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

PubMed Central

Occhipinti, Laura; Chang, Yiming; Altvater, Martin; Menet, Anna M.; Kemmler, Stefan; Panse, Vikram G.

2013-01-01

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel. PMID:23907389

A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

PubMed Central

Millard, S. Sean; Vidal, Anxo; Markus, Maurice; Koff, Andrew

2000-01-01

Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals. PMID:10913178

Interrelations between translation and general mRNA degradation in yeast.

PubMed

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. © 2014 The Authors. WIREs RNA published by John Wiley & Sons, Ltd.

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monaco, L.; Murtagh, J.J.; Newman, K.B.

1990-03-01

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and usedmore » to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.« less

Experiment K-6-11. Actin mRNA and cytochrome c mRNA concentrations in the tricepts brachia muscle of rats

NASA Technical Reports Server (NTRS)

Booth, F. W.; Morrison, P. R.; Thomason, D. B.; Oganov, V. S.

1990-01-01

It is well known that some skeletal muscles atrophy as a result of weightlessness (Steffen and Musacchia 1986) and as a result of hindlimb suspension (Tischler et al., 1985, Thomason et al., 1987). Because the content of protein is determined by the rates of protein synthesis and degradation, a decrease in protein synthesis rate, or an increase in the protein degradation, or changes in both could produce the atrophy. Indeed, an increased protein degradation (Tischler et al., 1985) and a decreased protein synthesis (Thomason et al., 1988) have been observed in skeletal muscles of suspended hindlimbs of rats. Any decrease in protein synthesis rate could be caused by decreases in mRNA concentrations. Such decreases in the concentration and content of alpha-actin mRNA and cytochrome c mRNA have been noted in skeletal muscles of hindlimb suspended rats (Babij and Booth, 1988). From these findings researchers hypothesized that alpha-actin mRNA and cytochrome c mRNA would decrease in the triceps brachia muscle of Cosmos 1887 rats.

Interrelations between translation and general mRNA degradation in yeast

PubMed Central

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. How to cite this article: WIREs RNA 2014, 5:747–763. doi: 10.1002/wrna.1244 PMID:24944158

Optimal Down Regulation of mRNA Translation

NASA Astrophysics Data System (ADS)

Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

2017-01-01

Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results.

Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

PubMed

Cho, Hana; Park, Ok Hyun; Park, Joori; Ryu, Incheol; Kim, Jeonghan; Ko, Jesang; Kim, Yoon Ki

2015-03-31

Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

The relative expression levels of insulin-like growth factor 1 and myostatin mRNA in the asynchronous development of skeletal muscle in ducks during early development.

PubMed

Hu, Yan; Liu, Hongxiang; Shan, Yanju; Ji, Gaige; Xu, Wenjuan; Shu, Jingting; Li, Huifang

2015-08-10

Genetic selection is a powerful tool for modifying poultry muscle yield. Insulin-like growth factor I (IGF-I) and myostatin (MSTN) are important regulators of muscle growth, especially in the myogenesis stage. This study compared the developmental pattern of the pectoralis major (PM) and lateral gastrocnemius (LM) muscles, mRNA expression characterization of IGF-I and MSTN-A and their correlation between 14 days in ovo and 1 week post-hatch in two Chinese local duck breeds. During early development, the growth of duck PM and LM followed an asynchronous pattern. Variations in PM growth rate observed with development followed the relative variations of MSTN and IGF-I expression; however, the same behavior was not observed in LM. Moreover, the profile of IGF-I expression in duck skeletal muscles indicated that genetic selection for high meat-yield poultry has altered the temporal expression of IGF-I and affected cellular characteristics and mass by hatch in a PM-specific manner. The MSTN-A expression profile showed synchronization with the growth of skeletal muscle and peaks of myofiber proliferation. The expression patterns of IGF-I and MSTN suggest that duck pectoralis fibers are prioritized for proliferation in embryogenesis. The IGF-1/MSTN-A mRNA ratios in PM and LM presented very similar trends in the changes of myofiber characteristics, and differences in the IGF-1/MSTN-A mRNA ratio in PM between the two breeds corresponded to the timing of differences in PM mass between the varieties. Our results support the hypothesis that relative levels of IGF-I and MSTN mRNA may participate in ordering muscle growth rates with selected development. Copyright © 2015 Elsevier B.V. All rights reserved.

Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemic rodent model.

PubMed

Shi, J; Yang, S H; Stubley, L; Day, A L; Simpkins, J W

2000-01-17

Silent stroke is one of the risk factors of dementia. In the present study, we used a novel focal ischemic animal model to investigate the effects of comparatively small changes of cerebral blood flow (CBF) on the expression of beta-amyloid precursor protein (APP) mRNA. Focal ischemia was achieved by introducing a 4-0 monofilament to the bifurcation of anterior and middle cerebral arteries. Brain samples were harvested from ischemic core and penumbra of cortices at 1, 4 and 7 days following ischemia. The expression of APP mRNA was assessed by RT-PCR. The CBF was decreased to 50% for 1 day after stroke and recovered to 90% at the fourth day after stroke. The changes of CBF were accompanied by an increase in the expression of APP mRNA. APP mRNA increased to 208% and 152% in the penumbra and core ischemic regions, respectively, on the fourth day after MCAO and remained high through the seventh day of ischemia. This study suggests brain hypoperfusion enhances APP mRNA expression and may contribute to the progression of cognitive impairment after silent stroke.

Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution

PubMed Central

Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

2013-01-01

Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the ‘long-term evolution experiment’. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins. PMID:23783573

Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration.

PubMed

Ziller, Antje; Nogueira, Sara S; Hühn, Eva; Funari, Sergio S; Brezesinski, Gerald; Hartmann, Hermann; Sahin, Ugur; Haas, Heinrich; Langguth, Peter

2018-02-05

Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

PubMed Central

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

2016-01-01

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

PubMed

Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

2016-03-01

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. © 2016 Müller-McNicoll et al.; Published by Cold Spring Harbor Laboratory Press.

Regulation of bone sialoprotein mRNA by steroid hormones

PubMed Central

1989-01-01

In this report we demonstrate an increase in the steady-state level of bone sialoprotein (BSP) mRNA in rat calvaria and a rat osteosarcoma cell line (ROS 17/2.8) after treatment with the synthetic glucocorticoid, dexamethasone. In contrast, 1.25-dihydroxyvitamin D3 reduced the amount of BSP mRNA in calvaria and inhibited the dexamethasone induction in ROS 17/2.8 cells. The increase in BSP mRNA is most likely due to an increase in the transcriptional rate. The stability of mRNA was unchanged after dexamethasone treatment with a half-life of approximately 5 h. Nuclear transcription experiments with nuclei isolated from ROS 17/2.8 cells showed an increased BSP mRNA synthesis in cells treated with dexamethasone. PMID:2592421

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

PubMed

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

In vivo characterization of the Drosophila mRNA 3′ end processing core cleavage complex

PubMed Central

Michalski, Daniel; Steiniger, Mindy

2015-01-01

A core cleavage complex (CCC) consisting of CPSF73, CPSF100, and Symplekin is required for cotranscriptional 3′ end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes nonpolyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. We used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272–1080 of Symplekin and the C-terminal approximately 200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3′ end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in dramatic increased use of downstream polyadenylation sites for histone mRNA 3′ end processing similar to RNAi-depletion of histone-specific 3′ end processing factors FLASH, SLBP, and U7 snRNA. We propose a model in which CCC formation is mediated by CPSF73, CPSF100, and Symplekin C-termini, and the N-terminal region of Symplekin facilitates cotranscriptional 3′ end processing of histone mRNAs. PMID:26081560

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.

PubMed

Gervasi, Noreen M; Scott, Shane S; Aschrafi, Armaz; Gale, Jenna; Vohra, Sanah N; MacGibeny, Margaret A; Kar, Amar N; Gioio, Anthony E; Kaplan, Barry B

2016-06-01

Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle.

PubMed

Shock, Jennifer L; Fischer, Kael F; DeRisi, Joseph L

2007-01-01

The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiologic role, and plays an important role in determining levels of gene expression. We used a genome-wide approach to characterize mRNA decay in Plasmodium falciparum. We found that, globally, rates of mRNA decay increase dramatically during the asexual intra-erythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 min, but this was extended to an average of 65 min during the late schizont stage of development. Thus, a major determinant of mRNA decay rate appears to be linked to the stage of intra-erythrocytic development. Furthermore, we found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intra-erythrocytic development. This type of genome-wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum.

Ways and means of eukaryotic mRNA decay.

PubMed

Balagopal, Vidya; Fluch, Lydia; Nissan, Tracy

2012-06-01

Messenger RNA degradation is an important point of control for gene expression. Genome-wide studies on mRNA stability have demonstrated its importance in adaptation and stress response. Most of the key players in mRNA decay appear to have been identified. The study of these proteins brings insight into the mechanism of general and specific targeting of transcripts for degradation. Recruitment and assembly of mRNP complexes enhance and bring specificity to mRNA decay. mRNP complexes can form larger structures that have been found to be ubiquitous in nature. Discovery of P-Bodies, an archetype of this sort of aggregates, has generated interest in the question of where mRNA degrades. This is currently an open question under extensive investigation. This review will discuss in detail the recent developments in the regulation of mRNA decay focusing on yeast as a model system. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing. Copyright © 2012 Elsevier B.V. All rights reserved.

Protamine mRNA ratio in stallion spermatozoa correlates with mare fecundity.

PubMed

Paradowska-Dogan, A; Fernandez, A; Bergmann, M; Kretzer, K; Mallidis, C; Vieweg, M; Waliszewski, P; Zitzmann, M; Weidner, W; Steger, K; Kliesch, S

2014-07-01

Highly compacted sperm DNA in protamine toroids and a minor fraction of nucleohistones are prerequisites for the efficient transmission of the paternal genome into the oocyte at fertilization. The objective of this study was to evaluate whether protamines might serve as a prognostic factor for stallion fertility. In situ hybridization detected specific expression of P1 mRNA in the cytoplasm of stage I to VII spermatids, whereas comparable immunohistochemical stainings showed that protein expression was delayed till elongating spermatids in differentiation stages III to VIII. No staining was detectable in cryptorchid testis because of the lack of spermatids in the seminiferous tubules. Using quantitative real-time polymerase chain reaction, we identified mRNA transcripts of P1 and 2 variants of protamine- 2 (P2, P3) in ejaculated spermatozoa from 45 thoroughbred stallions. According to the mare fertility descriptor (i.e. the 'none-return-rate 28 percentage' or NRR28%), stallions were divided into three groups (i.e. high, reduced and low fertility). The P2/P1 mRNA ratio was found to be significantly reduced in the group with lower fertility (p = 0.016) and was slightly correlated with sperm concentration (correlation coefficient r = 0.263). Furthermore, morphologically abnormal sperm count negatively correlated with P2/P1 mRNA ratio, indicating that spermatozoa carrying head defects display a diminished protamine ratio (r = -0.348). Conversely, the P2/P1 ratio was positively correlated with mare fertility or NRR28% (r = 0.274). Interestingly, P3/P1 mRNA ratio remained unaltered in the investigated groups indicating that this variant plays a minor role in equine sperm chromatin compaction. Aberrant protamine transcripts content in equine spermatozoa was not associated with DNA defragmentation rate as measured by flow cytometric acridine orange test. On the basis of these results, we suggest that, similar to human, equine protamine expression constitutes a checkpoint

Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones.

PubMed

Kononen, J; Soinila, S; Persson, H; Honkaniemi, J; Hökfelt, T; Pelto-Huikko, M

1994-12-01

We studied the expression of messenger ribonucleic acids (mRNAs) for neurotrophins and neurotrophin receptors in the rat pituitary gland and examined the influence of adrenal hormones on their mRNA levels, using in situ hybridization and Northern blot analysis. The only neurotrophin present at detectable levels in the pituitary was brain-derived neurotrophic factor (BDNF), which was observed in the anterior and intermediate lobes. Several transcripts of the putative receptor for BDNF, trkB, were present in the anterior and posterior lobes of the pituitary. A low amount of trkC mRNA was found in both the anterior and the intermediate lobe. Dexamethasone treatment decreased both BDNF and trkB mRNA levels in the anterior lobe of the pituitary. Adrenalectomy had no effect on trkB expression, but it decreased BDNF mRNA levels in comparison to the control animals. This effect could not be reversed by dexamethasone substitution, suggesting that BDNF, mRNA levels may be regulated not only by glucocorticoids but also by other adrenal hormones. These results demonstrate that BDNF, trkB and trkC are expressed in the pituitary gland and that glucocorticoids and possibly other adrenal hormones may modulate pituitary functions by regulating the expression of neurotrophic factors and their receptors. Whether BDNF acts as a secreted hormone, a trophic factor, or has autocrine/paracrine functions within the pituitary through its receptor, trkB, remains to be studied.

Patterns of mRNA and protein expression during minus-lens compensation and recovery in tree shrew sclera.

PubMed

Gao, Hong; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2011-04-12

To increase our understanding of the mechanisms that remodel the sclera during the development of lens-induced myopia, when the sclera responds to putative "go" signals of retinal origin, and during recovery from lens-induced myopia, when the sclera responds to retinally-derived "stop" signals. Seven groups of tree shrews were used to examine mRNA levels during minus lens compensation and recovery. Starting 24 days after eye opening (days of visual experience [VE]) lens compensation animals wore a monocular -5D lens for 1, 4, or 11 days. Recovery animals wore the -5D lens for 11 days, which was then removed for 1 or 4 days. Normal animals were examined at 24 and 38 days of VE. All groups contained 8 animals. Scleral mRNA levels were examined in the treated and contralateral control eyes with quantitative real-time polymerase chain reaction (qPCR) for 27 genes divided into four categories: 1) signaling molecules, 2) matricellular proteins, 3) metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs), and 4) cell adhesion and other proteins. Four groups (n=5 per group) were used to examine protein levels. One group wore a -5D lens for 4 days. A second group recovered for 4 days after 11 days of -5D lens treatment. Two groups were used to examine age-matched normal protein levels at 28 and 39 days of VE. The levels of six scleral proteins that showed differential mRNA expression were examined with quantitative western blots. Nineteen of the genes showed differential (treated eye versus control eye) expression of mRNA levels in at least one group of animals. Which genes showed differential expression differed after 1 and 4 days of compensation and after 1 or 4 days of recovery. The mRNA level for one gene, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), was upregulated in the treated eyes after 1 day of compensation. After 4 days, transforming growth factor beta receptor 3 (TGFBR3), transforming growth factor

Activation of Tyrosine Hydroxylase mRNA Translation by cAMP in Midbrain Dopaminergic Neurons

PubMed Central

Chen, Xiqun; Xu, Lu; Radcliffe, Pheona; Sun, Baoyong; Tank, A. William

2009-01-01

During prolonged stress or chronic treatment with neurotoxins, robust compensatory mechanisms occur which maintain sufficient levels of catecholamine neurotransmitters in terminal regions. One of these mechanisms is the up-regulation of tyrosine hydroxylase (TH), the enzyme that controls catecholamine biosynthesis. In neurons of the periphery and locus coeruleus, this up-regulation is associated with an initial induction of TH mRNA. In contrast, this induction either does not occur or is nominal in mesencephalic dopamine neurons. The reasons for this lack of compensatory TH mRNA induction remain obscure, because so little is known about the regulation of TH expression in these neurons. In this report we test whether activation of the cAMP signaling pathway regulates TH gene expression in two rodent models of midbrain dopamine neurons, ventral midbrain organotypic slice cultures and MN9D cells. Our results demonstrate that elevation of cAMP leads to induction of TH protein and TH activity in both model systems; however, TH mRNA levels are not up-regulated by cAMP. The induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH mRNA translation. This translational activation is mediated by sequences within the 3′UTR of TH mRNA. Our results support a model in which cAMP induces or activates trans-factors that interact with the TH mRNA 3′UTR to increase TH protein synthesis. An understanding of this novel regulatory mechanism may help to explain the control of TH gene expression and consequently dopamine biosynthesis in midbrain neurons under different physiological and pathological conditions. PMID:18349104

The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA.

PubMed

Di Santo, Rachael; Aboulhouda, Soufiane; Weinberg, David E

2016-10-01

HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation-both dependent on the intron-prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA.

Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder (Paralichthys olivaceus).

PubMed

Zhang, Junling; Shi, Zhiyi; Cheng, Qi; Chen, Xiaowu

2011-08-01

Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial-temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder. Copyright © 2011 Elsevier Inc. All rights reserved.

Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

NASA Astrophysics Data System (ADS)

Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

1988-12-01

Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

PubMed

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

2016-04-20

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Changes in the mRNA levels of delayed rectifier potassium channels in human atrial fibrillation.

PubMed

Lai, L P; Su, M J; Lin, J L; Lin, F Y; Tsai, C H; Chen, Y S; Tseng, Y Z; Lien, W P; Huang, S K

1999-01-01

We measured mRNA levels of delayed rectifier potassium channels in human atrial tissue to investigate the mechanism of the shortening of the atrial effective refractory period and the loss of rate-adaptive shortening of the atrial effective refractory period in human atrial fibrillation. A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The mRNA amounts of KVLQT1 (IKs), minK (beta-subunit of IKs), HERG (IKr), and KV1.5 (IKur) were measured by reverse transcription-polymerase chain reaction and normalized to the mRNA amount of GAPDH. We found that the mRNA levels of KV1.5, HERG and KVLQT1 were all significantly decreased in patients with persistent atrial fibrillation for more than 3 months. In contrast, the mRNA level of minK was significantly increased in patients with persistent atrial fibrillation for more than 3 months. We further showed that these changes were independent of the underlying cardiac disease, atrial filling pressure, gender and age. We also found that there was no spatial dispersion of mRNA levels among the four atrial sampling sites. Because the decrease in potassium currents results in a prolonged action potential, the shortening of the atrial effective refractory period in atrial fibrillation should be attributed to other factors. However, the decrease in IKs might contribute, at least in part, to the loss of rate-adaptive shortening of the atrial refractory period.

Rational Manipulation of mRNA Folding Free Energy Allows Rheostat Control of Pneumolysin Production by Streptococcus pneumoniae

PubMed Central

Amaral, Fábio E.; Parker, Dane; Randis, Tara M.; Kulkarni, Ritwij; Prince, Alice S.; Shirasu-Hiza, Mimi M.; Ratner, Adam J.

2015-01-01

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic “knockouts” that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5’ end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5’ mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5’ end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis. PMID:25798590

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma Irradiation

DTIC Science & Technology

2014-12-01

function of miRNAs. For example, TTP promotes tumor ne- crosis factor alpha (TNF-") mRNA decay caused by miR-16 (29) and HuR facilitates the targeting of c...experiments would demonstrate an important function of L11 in regulating c-myc mRNA in response to DNA damage, offer useful information for developing...24 (Figure 1). We also tested an array of miRNAs possessing tumor suppressor functions for L11 binding miR-16 miR-1248 miR-3944 (-) miR-191 miR

mRNA vaccines — a new era in vaccinology

PubMed Central

Pardi, Norbert; Hogan, Michael J.; Porter, Frederick W.; Weissman, Drew

2018-01-01

mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use. PMID:29326426

Effects of retinoids and thiazolidinediones on proliferation, insulin release, insulin mRNA, GLUT 2 transporter protein and mRNA of INS-1 cells.

PubMed

Blumentrath, J; Neye, H; Verspohl, E J

2001-09-01

Both 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (ATRA) are active metabolites of vitamin A (retinol). There exists an interaction between retinoid receptors and peroxisome proliferator-activated receptors (PPARgamma). To define their functions in an insulin secreting system the effects of ATRA, 9cRA and the PPARgamma agonist rosiglitazone on cell proliferation, insulin release and glucose transporter (GLUT) 2 of INS-1 cells were tested. Retinoic acid receptor (RAR-alpha and -gamma) and retinoid X receptor (RXR-alpha and -beta) proteins are present (immunoblots). Both 9cRA and ATRA inhibit INS-1 cell proliferation ([3H]-thymidine assay) in a concentration dependent manner. Both 9cRA and ATRA increased insulin release, but only ATRA ralsed the GLUT 2 mRNA in a bell-shaped concentration response curve after 48 h. The insulinotropic effect of one compound is not significantly superimposed by the other indicating that the same binding sites are used by 9cRA and ATRA. The acute and chronic effects of the PPARgamma agonist rosiglitazone on insulin release were additionally determined since glitazones act as transcription factors together with RXR agonists. At high concentrations (100 microM) rosiglitazone inhibited glucose (8.3 mM) stimulated insulin secretion (acute experiment over 60 min). Insulin secretion, however, was increased during a 24 h treatment at a concentration of 10 microM and again inhibited at 100 microM. Changes in preproinsulin mRNA expression were not observed. Rosiglitazone (100 microM) increased GLUT 2 mRNA paralleled by an increase of GLUT 2 protein, but only after 24 h of treatment. This data indicate that RAR and RXR mediate insulin release. The changes in GLUT 2 have no direct impact on insulin release; the inhibition seen at high concentrations of either compound is possibly the result of the observed inhibition of cell proliferation. Effects of rosiglitazone on preproinsulin mRNA and GLUT 2 (mRNA and protein) do not play a role in

An ‘environment to nucleus’ signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation

PubMed Central

Tell, Gianluca; Zecca, Alessandro; Pellizzari, Lucia; Spessotto, Paola; Colombatti, Alfonso; Kelley, Mark R.; Damante, Giuseppe; Pucillo, Carlo

2000-01-01

The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H2O2 induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse–chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells. PMID:10666449

Ventilation-induced increases in EGFR ligand mRNA are not altered by intra-amniotic LPS or ureaplasma in preterm lambs.

PubMed

Hillman, Noah H; Gisslen, Tate; Polglase, Graeme R; Kallapur, Suhas G; Jobe, Alan H

2014-01-01

Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.

Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

PubMed

Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

2014-09-01

The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis

[Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

PubMed

Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

2009-06-01

To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

PubMed Central

Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

2016-01-01

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

PubMed Central

Posternak, Valeriya; Ung, Matthew H.; Cheng, Chao; Cole, Michael D.

2016-01-01

MYC is a pleiotropic transcription factor that activates and represses a wide range of target genes and is frequently deregulated in human tumors. While much is known about the role of MYC in transcriptional activation and repression, MYC can also regulate mRNA cap methylation through a mechanism that has remained poorly understood. Here it is reported that MYC enhances mRNA cap methylation of transcripts globally, specifically increasing mRNA cap methylation of genes involved in Wnt/β-catenin signaling. Elevated mRNA cap methylation of Wnt signaling transcripts in response to MYC leads to augmented translational capacity, elevated protein levels, and enhanced Wnt signaling activity. Mechanistic evidence indicates that MYC promotes recruitment of RNA methyltransferase (RNMT) to Wnt signaling gene promoters by enhancing phosphorylation of serine 5 on the RNA Polymerase II Carboxy-Terminal Domain, mediated in part through an interaction between the TIP60 acetyltransferase complex and TFIIH. Implications MYC enhances mRNA cap methylation above and beyond transcriptional induction. PMID:27899423

Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maquat, Lynne

2002-12-01

The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryoticmore » nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?« less

APP mRNA splicing is upregulated in the brain of biglycan transgenic mice.

PubMed

Bjelik, Annamária; Pákáski, Magdolna; Bereczki, Erika; Gonda, Szilvia; Juhász, Anna; Rimanóczy, Agnes; Zana, Marianna; Janka, Zoltán; Sántha, Miklós; Kálmán, János

2007-01-01

Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of beta-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB(+/-)xbiglycan(+/-)) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.

Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats.

PubMed

Silverstein, J H; Beasley, J; Mizuno, T M; London, E; Mobbs, C V

1998-04-01

Relatively few molecular markers of stress have been studied in aged individuals. Interactions of age and stress on adrenal neuropeptide Y (NPY) and preproenkephalin (ppENK) expression have not been reported. The purpose of these studies was to characterize the adrenal NPY and ppENK responses to stress using a common stressor, physical restraint for 2 h, in Fischer 344 rats at 7, 16 and 23 months of age. Northern blot techniques were used to evaluate induction by stress of adrenal NPY mRNA and adrenal ppENK mRNA. Two humoral responses to stress, serum glucose and corticosterone, were measured to corroborate that a stress response occurred. We observed that the induction by stress of adrenal NPY mRNA is impaired with age but the stress-induced elevation of adrenal ppENK mRNA, blood glucose, and corticosterone show no evidence of age-related impairments.

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3.

PubMed

De, B P; Galinski, M S; Banerjee, A K

1990-03-01

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.

PubMed

O'Leary, Nuala A; Wright, Mathew W; Brister, J Rodney; Ciufo, Stacy; Haddad, Diana; McVeigh, Rich; Rajput, Bhanu; Robbertse, Barbara; Smith-White, Brian; Ako-Adjei, Danso; Astashyn, Alexander; Badretdin, Azat; Bao, Yiming; Blinkova, Olga; Brover, Vyacheslav; Chetvernin, Vyacheslav; Choi, Jinna; Cox, Eric; Ermolaeva, Olga; Farrell, Catherine M; Goldfarb, Tamara; Gupta, Tripti; Haft, Daniel; Hatcher, Eneida; Hlavina, Wratko; Joardar, Vinita S; Kodali, Vamsi K; Li, Wenjun; Maglott, Donna; Masterson, Patrick; McGarvey, Kelly M; Murphy, Michael R; O'Neill, Kathleen; Pujar, Shashikant; Rangwala, Sanjida H; Rausch, Daniel; Riddick, Lillian D; Schoch, Conrad; Shkeda, Andrei; Storz, Susan S; Sun, Hanzhen; Thibaud-Nissen, Francoise; Tolstoy, Igor; Tully, Raymond E; Vatsan, Anjana R; Wallin, Craig; Webb, David; Wu, Wendy; Landrum, Melissa J; Kimchi, Avi; Tatusova, Tatiana; DiCuccio, Michael; Kitts, Paul; Murphy, Terence D; Pruitt, Kim D

2016-01-04

The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55,000 organisms (>4800 viruses, >40,000 prokaryotes and >10,000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

PubMed Central

Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

2014-01-01

Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

PKA- and PKC-dependent regulation of angiopoietin 2 mRNA in human granulosa lutein cells.

PubMed

Witt, P S; Pietrowski, D; Keck, C

2004-02-01

New blood vessels develop from preexisting vessels in response to growth factors or hypoxic conditions. Recent studies have shown that angiopoietin 2 (ANGPT-2) plays an important role in the modulation of angiogenesis and vasculogenesis in humans and mice. The signaling pathways that lead to the regulation of ANGPT-2 are largely unclear. Here, we report that protein kinase C and protein kinase A activators (ADMB, 8-Cl-cAMP) increased the mRNA levels of ANGPT-2 in human Granulosa cells, whereas PKC and PKA Inhibitors (Rp-cAMP, GO 6983) decreased markedly the level of ANGPT-2 mRNA. Due to varying specificity of the modulators for certain protein kinases subunits, we conclude that the conventional PKCs, but not PKC alpha and beta1, the atypical PKCs and the PKA I, are involved in the regulation of ANGPT-2. These findings may help to explain the role of both PKA and PKC dependent signaling cascades in the regulation of ANGPT-2 mRNA.

A mRNA and cognate microRNAs localize in the nucleolus.

PubMed

Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

2014-01-01

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Substance P induced preprotachykinin-a mRNA, neutral endopeptidase mRNA and substance P in cultured normal fibroblasts.

PubMed

Bae, Sang-Jae; Matsunaga, Yoshitaka; Takenaka, Motoi; Tanaka, Yoichi; Hamazaki, Yoichiro; Shimizu, Kazuhiro; Katayama, Ichiro

2002-04-01

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1-3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30-40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases. Copyright 2002 S. Karger AG, Basel

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans

PubMed Central

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-01-01

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. PMID:22508728

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

PubMed

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-04-15

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Tools for translation: non-viral materials for therapeutic mRNA delivery

NASA Astrophysics Data System (ADS)

Hajj, Khalid A.; Whitehead, Kathryn A.

2017-10-01

In recent years, messenger RNA (mRNA) has come into the spotlight as a versatile therapeutic with the potential to prevent and treat a staggering range of diseases. Billions of dollars have been invested in the commercial development of mRNA drugs, with ongoing clinical trials focused on vaccines (for example, influenza and Zika viruses) and cancer immunotherapy (for example, myeloma, leukaemia and glioblastoma). Although significant progress has been made in the design of in vitro-transcribed mRNA that retains potency while minimizing unwanted immune responses, the widespread use of mRNA drugs requires the development of safe and effective drug delivery vehicles. In this Review, we provide an overview of the field of mRNA therapeutics and describe recent advances in the development of synthetic materials that encapsulate and deliver mRNA payloads.

Connections Underlying Translation and mRNA Stability.

PubMed

Radhakrishnan, Aditya; Green, Rachel

2016-09-11

Gene expression and regulation in organisms minimally depends on transcription by RNA polymerase and on the stability of the RNA product (for both coding and non-coding RNAs). For coding RNAs, gene expression is further influenced by the amount of translation by the ribosome and by the stability of the protein product. The stabilities of these two classes of RNA, non-coding and coding, vary considerably: tRNAs and rRNAs tend to be long lived while mRNAs tend to be more short lived. Even among mRNAs, however, there is a considerable range in stability (ranging from seconds to hours in bacteria and up to days in metazoans), suggesting a significant role for stability in the regulation of gene expression. Here, we review recent experiments from bacteria, yeast and metazoans indicating that the stability of most mRNAs is broadly impacted by the actions of ribosomes that translate them. Ribosomal recognition of defective mRNAs triggers "mRNA surveillance" pathways that target the mRNA for degradation [Shoemaker and Green (2012) ]. More generally, even the stability of perfectly functional mRNAs appears to be dictated by overall rates of translation by the ribosome [Herrick et al. (1990), Presnyak et al. (2015) ]. Given that mRNAs are synthesized for the purpose of being translated into proteins, it is reassuring that such intimate connections between mRNA and the ribosome can drive biological regulation. In closing, we consider the likelihood that these connections between protein synthesis and mRNA stability are widespread or whether other modes of regulation dominate the mRNA stability landscape in higher organisms. Copyright © 2016. Published by Elsevier Ltd.

The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events.

PubMed

Sørensen, Brian B; Ehrnsberger, Hans F; Esposito, Silvia; Pfab, Alexander; Bruckmann, Astrid; Hauptmann, Judith; Meister, Gunter; Merkl, Rainer; Schubert, Thomas; Längst, Gernot; Melzer, Michael; Grasser, Marion; Grasser, Klaus D

2017-02-01

We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

PubMed

Fonseca, Bruno D; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M; Diao, Ilo T; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L; Hernández, Greco; Alain, Tommy; Damgaard, Christian K

2015-06-26

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1)*

PubMed Central

Fonseca, Bruno D.; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E.; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M.; Diao, Ilo T.; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M.; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L.; Hernández, Greco; Alain, Tommy; Damgaard, Christian K.

2015-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. PMID:25940091

Coupling mRNA processing with transcription in time and space

PubMed Central

Bentley, David L.

2015-01-01

Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3′ end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes. PMID:24514444

Macrophages from Behcet's Disease Patients Express Decreased Level of Aryl Hydrocarbon Receptor (AHR) mRNA.

PubMed

Palizgir, Mohammad Taghi; Akhtari, Maryam; Mahmoudi, Mahdi; Mostafaei, Shayan; Rezaeimanesh, Alireza; Akhlaghi, Massoomeh; Shahram, Farhad

2017-10-01

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, connecting environmental stimulators with the immune system. M1 macrophages are a part of immune system that contribute to the inflammatory events in the pathogenesis of Behcet's disease (BD). The effect of AHR on the macrophages in BD patients is still unclear. In this study, we investigated the mRNA expression of AHR in the monocyte-derived and M1 macrophages in active BD patients in comparison to healthy controls. Isolated monocytes from 10 healthy controls and 10 active BD patients were differentiated to macrophages by macrophage-colony stimulating factor (M-CSF) for 7 days. Cells were then polarized to M1 macrophages by lipopolysaccharide (LPS) and interferon-γ (IFNγ) for 24h. Monocyte purity and macrophage markers expression were analyzed by flow cytometry. Analysis of AHR mRNA expression was performed by SYBR Green real-time PCR. Our results showed that AHR expression is significantly down-regulated in M1 macrophages compare to monocyte-derived macrophages. It was shown that both monocyte-derived macrophages and M1 macrophages from BD patients significantly express lower level of AHR mRNA compared to healthy individuals. Our results demonstrate an anti-inflammatory role for AHR in macrophages, which suggest that decreased AHR expression is associated with pro-inflammatory M1 macrophage and BD susceptibility.

Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin.

PubMed

Thomsen, Rune; Pallesen, Jonatan; Daugaard, Tina F; Børglum, Anders D; Nielsen, Anders L

2013-11-01

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance. Copyright © 2013 Wiley Periodicals, Inc.

Expression of beta 3-adrenoceptor mRNA in rat tissues.

PubMed

Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

1996-01-01

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with

SEIZURE ACTIVITY INVOLVED IN THE UP-REGULATION OF BDNF mRNA EXPRESSION BY ACTIVATION OF CENTRAL MU OPIOID RECEPTORS

PubMed Central

ZHANG, H. N.; KO, M. C.

2009-01-01

Chemical-induced seizures up-regulated brain-derived neurotrophic factor (BDNF) mRNA expression. Intracerebroventricular (i.c.v.) administration of endogenous opioids preferentially activating μ opioid receptor (MOR) could also increase BDNF mRNA expression. The aim of this study was to determine to what extent i.c.v. administration of synthetic MOR-selective agonists in rats can modulate both seizure activity and up-regulation of BDNF mRNA expression. Effects and potencies of i.c.v. administration of morphine and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), were directly investigated by scoring behavioral seizures and measuring BDNF mRNA expression. In addition, effects of the opioid receptor antagonist naloxone and antiepileptic drugs, diazepam, phenobarbital, and valproate, on i.c.v. MOR agonist-induced behavioral seizures and up-regulation of BDNF mRNA expression were determined. A single i.c.v. administration of morphine (10–100 μg) or DAMGO (0.15–1.5 μg) dose-dependently elicited behavioral seizures and increased BDNF mRNA expression in the widespread brain regions. However, subcutaneous administration of MOR agonists neither produced behavioral seizures nor increased BDNF mRNA expression. Pretreatment with naloxone 1 mg/kg significantly reduced behavioral seizure scores and the up-regulation of BDNF mRNA expression elicited by i.c.v. morphine or DAMGO. Similarly, diazepam 10 mg/kg and phenobarbital 40 mg/kg significantly blocked i.c.v. MOR agonist-induced actions. Pretreatment with valproate 300 mg/kg only attenuated behavioral seizures, but it did not affect morphine-induced increase of BDNF mRNA expression. This study provides supporting evidence that seizure activity plays an important role in the up-regulation of BDNF mRNA expression elicited by central MOR activation and that decreased inhibitory action of GABAergic system through the modulation on GABA receptor synaptic function by central MOR activation is involved in its regulation of BDNF mRNA

Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas.

PubMed

Chen, Xiaochuan; Rozance, Paul J; Hay, William W; Limesand, Sean W

2012-05-01

Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

PubMed

Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

2016-01-01

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

mRNA transcripts as molecular biomarkers in medicine and nutrition

PubMed Central

Sunde, Roger A.

2010-01-01

In medicine, mRNA transcripts are being developed as molecular biomarkers for the diagnosis and treatment of a number of diseases. These biomarkers offer early and more accurate prediction and diagnosis of disease and disease progression, and ability to identify individuals at risk. Use of microarrays also offers opportunity to identify orthogonal (uncorrelated) biomarkers not known to be linked with conventional biomarkers. Investigators are increasingly using blood as a surrogate tissue for biopsy and analysis; total RNA isolated from whole blood is predominantly from erythroid cells, and whole blood mRNA share more than 80% of the transcriptome with major tissues. Thus blood mRNA biomarkers for individualized disease prediction and diagnosis are an exciting area in medicine; mRNA biomarkers in nutrition have potential application that parallel these opportunities. Assessment of selenium (Se) status and requirements is one area where tissue mRNA levels have been used successfully. Selenoprotein-H and selenoprotein-W as well as glutathione peroxidase-1 (Gpx1) mRNAs are highly down-regulated in Se deficiency in rat liver, and the minimum dietary Se requirement is 0.06–0.07 μg Se/g based on these biomarkers, similar to requirements determined using conventional biomarkers. Blood Gpx1 mRNA can also be used to determine Se requirements in rats, showing that blood mRNA has potential for assessment of nutrient status. Future research is needed to develop mRNA biomarker panels for all nutrients that will discriminate between deficient, marginal, adequate, and supernutritional individuals and populations, and differentiate between individuals that will benefit versus be adversely affected by nutrient supplementation. PMID:20303730

mRNA localization: an orchestration of assembly, traffic and synthesis.

PubMed

Xing, Lei; Bassell, Gary J

2013-01-01

Asymmetrical mRNA localization and subsequent local translation provide efficient mechanisms for protein sorting in polarized cells. Defects in mRNA localization have been linked to developmental abnormalities and neurological diseases. Thus, it is critical to understand the machineries mediating and mechanisms underlying the asymmetrical distribution of mRNA and its regulation. The goal of this review is to summarize recent advances in the understanding of mRNA transport and localization, including the assembly and sorting of transport messenger ribonucleic protein (mRNP) granules, molecular mechanisms of active mRNP transport, cytoskeletal interactions and regulation of these events by extracellular signals. © 2012 John Wiley & Sons A/S.

Macrophage migration inhibitory factor as an incriminating agent in vitiligo.

PubMed

Farag, Azza Gaber Antar; Hammam, Mostafa Ahmed; Habib, Mona SalahEldeen; Elnaidany, Nada Farag; Kamh, Mona Eaid

2018-03-01

Vitiligo is an autoimmune skin disorder in which the loss of melanocytes is mainly attributed to defective autoimmune mechanisms and, lately, there has been more emphasis on autoinflammatory mediators. Among these is the macrophage migration inhibitory factor, which is involved in many autoimmune skin diseases. However, little is known about the contribution of this factor to vitiligo vulgaris. To determine the hypothesized role of migration inhibitory factor in vitiligo via estimation of serum migration inhibitory factor levels and migration inhibitory factor mRNA concentrations in patients with vitiligo compared with healthy controls. We also aimed to assess whether there is a relationship between the values of serum migration inhibitory factor and/or migration inhibitory factor mRNA with disease duration, clinical type and severity in vitiligo patients. Evaluation of migration inhibitory factor serum level and migration inhibitory factor mRNA expression by ELISA and real-time PCR, respectively, were performed for 50 patients with different degrees of vitiligo severity and compared to 15 age- and gender-matched healthy volunteers as controls. There was a highly significant increase in serum migration inhibitory factor and migration inhibitory factor mRNA levels in vitiligo cases when compared to controls (p<0.001). There was a significant positive correlation between both serum migration inhibitory factor and migration inhibitory factor mRNA concentrations in vitiligo patients, and each of them with duration and severity of vitiligo. In addition, patients with generalized vitiligo have significantly elevated serum migration inhibitory factor and mRNA levels than control subjects. Small number of investigated subjects. Migration inhibitory factor may have an active role in the development of vitiligo, and it may also be a useful index of disease severity. Consequently, migration inhibitory factor may be a new treatment target for vitiligo patients.

Differential utilization of decapping enzymes in mammalian mRNA decay pathways

PubMed Central

Li, You; Song, Mangen; Kiledjian, Megerditch

2011-01-01

mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells. PMID:21224379

IL-4 mRNA Is Downregulated in the Liver of Pancreatic Cancer Patients Suffering from Cachexia.

PubMed

Prokopchuk, Olga; Steinacker, Jürgen M; Nitsche, Ulrich; Otto, Stephanie; Bachmann, Jeannine; Schubert, Elaine C; Friess, Helmut; Martignoni, Marc E

2017-01-01

Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia. Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1. We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness. We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

PubMed Central

Pasquinelli, Amy E.; Powers, Maureen A.; Lund, Elsebet; Forbes, Douglass; Dahlberg, James E.

1997-01-01

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery. PMID:9405623

Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus.

PubMed

Kasimanickam, Ramanathan K; Kasimanickam, Vanmathy R; Haldorson, Gary J; Tibary, Ahmed

2012-01-23

Interleukins (IL) play an important role in angiogenesis. Tocopherol possesses immunomodulating effect in addition to antioxidant property. The objective of this study was to determine whether gamma tocopherol's (gT) angiogenic activity in placental network is enhanced via promoting interleukins. Pregnant ewes (N=18) were supplemented, orally, with 500 mg of alpha tocopherol (aT; N=6) or 1,000 mg of gT (N=7) or placebo (CON; N=5) once daily from 107 to 137 days post breeding. Uterine and placental tissue samples were obtained at the end of supplementation to evaluate relative mRNA expressions of IL-1b, IL-6, IL-8, Tumor Necrosis Factor (TNF) alpha, Vascular Endothelial Growth Factor (VEGF), kinase insert domain receptor (KDR; VGFR2; a type III receptor tyrosine kinase), and soluble fms-like tyrosine kniase-1 (sFlt1 or sVEGFR1) in uterus, caruncle and cotyledon. Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA abundances whereas sFlt1 mRNA abundance was suppressed in uterus, caruncle and cotyledon, compared to aT and placebo treated ewes (P<0.05). The TNF alpha and IL-1b mRNA abundances were suppressed in uterus, caruncle and cotyledon but TNF alpha is higher in gT group compared to aT group (P<0.05), whereas IL-1b was similar between treatment groups (P>0.1). Gamma tocopherol supplementation increased IL-6, IL-8, and KDR mRNA abundances and suppressed sFlt1 and TNFalpha mRNA abundances thereby increased VEGF mRNA expression and angiogenesis in placental vascular network during late gestation. It is plausible that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8.

Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus

PubMed Central

2012-01-01

Background Interleukins (IL) play an important role in angiogenesis. Tocopherol possesses immunomodulating effect in addition to antioxidant property. The objective of this study was to determine whether gamma tocopherol's (gT) angiogenic activity in placental network is enhanced via promoting interleukins. Methods Pregnant ewes (N = 18) were supplemented, orally, with 500 mg of alpha tocopherol (aT; N = 6) or 1,000 mg of gT (N = 7) or placebo (CON; N = 5) once daily from 107 to 137 days post breeding. Uterine and placental tissue samples were obtained at the end of supplementation to evaluate relative mRNA expressions of IL-1b, IL-6, IL-8, Tumor Necrosis Factor (TNF) alpha, Vascular Endothelial Growth Factor (VEGF), kinase insert domain receptor (KDR; VGFR2; a type III receptor tyrosine kinase), and soluble fms-like tyrosine kniase-1 (sFlt1 or sVEGFR1) in uterus, caruncle and cotyledon. Results Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA abundances whereas sFlt1 mRNA abundance was suppressed in uterus, caruncle and cotyledon, compared to aT and placebo treated ewes (P < 0.05). The TNF alpha and IL-1b mRNA abundances were suppressed in uterus, caruncle and cotyledon but TNF alpha is higher in gT group compared to aT group (P < 0.05), whereas IL-1b was similar between treatment groups (P > 0.1). Conclusions Gamma tocopherol supplementation increased IL-6, IL-8, and KDR mRNA abundances and suppressed sFlt1 and TNFalpha mRNA abundances thereby increased VEGF mRNA expression and angiogenesis in placental vascular network during late gestation. It is plausible that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8. PMID:22269218

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential

PubMed Central

Strenkowska, Malwina; Grzela, Renata; Majewski, Maciej; Wnek, Katarzyna; Kowalska, Joanna; Lukaszewicz, Maciej; Zuberek, Joanna; Darzynkiewicz, Edward; Kuhn, Andreas N.; Sahin, Ugur; Jemielity, Jacek

2016-01-01

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5′ end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization. PMID:27903882

HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA.

PubMed

Ren, Xiao-Xin; Wang, Hai-Bo; Li, Chuan; Jiang, Jin-Feng; Xiong, Si-Dong; Jin, Xia; Wu, Li; Wang, Jian-Hua

2016-02-26

HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Adverse early life experience and social stress during adulthood interact to increase serotonin transporter mRNA expression

PubMed Central

Gardner, Katherine L.; Hale, Matthew W.; Lightman, Stafford L.; Plotsky, Paul M.; Lowry, Christopher A.

2009-01-01

Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of serotonergic systems in the brain. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on serotonin transporter (slc6a4) mRNA expression, we investigated in rats the effects of maternal separation (180 min/day from days 2–14 of life; MS180), neonatal handing (15 min/day from days 2–14 of life; MS15), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on slc6a4 mRNA expression in the dorsal raphe nucleus (DR) and caudal linear nucleus. At the level of specific subdivisions of the DR, there were no differences in slc6a4 mRNA expression between MS15 and AFR rats. Among rats exposed to a novel cage control condition, increased slc6a4 mRNA expression was observed in the dorsal part of the DR in MS180 rats, relative to AFR control rats. In contrast, MS180 rats exposed to social defeat as adults had increased slc6a4 mRNA expression throughout the DR compared to both MS15 and AFR controls. Social defeat increased slc6a4 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR. Overall these data demonstrate that early life experience and stressful experience during adulthood interact to determine slc6a4 mRNA expression. These data support the hypothesis that early life experience and major stressful life events contribute to dysregulation of serotonergic systems in stress-related neuropsychiatric disorders. PMID:19781533

mRNA interactome capture in mammalian cells.

PubMed

Kastelic, Nicolai; Landthaler, Markus

2017-08-15

Throughout their entire life cycle, mRNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions. Their interplay is one key to control gene regulatory mechanisms from mRNA synthesis to decay. To assay the global scope of RNA-protein interactions, we and others have published a method combining crosslinking with highly stringent oligo(dT) affinity purification to enrich proteins associated with polyadenylated RNA (poly(A)+ RNA). Identification of the poly(A)+ RNA-bound proteome (also: mRNA interactome capture) has by now been applied to a diversity of cell lines and model organisms, uncovering comprehensive repertoires of RBPs and hundreds of novel RBP candidates. In addition to determining the RBP catalog in a given biological system, mRNA interactome capture allows the examination of changes in protein-mRNA interactions in response to internal and external stimuli, altered cellular programs and disease. Copyright © 2017. Published by Elsevier Inc.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.

PubMed

Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric

2016-01-01

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

PubMed

Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

PubMed

Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

2016-01-01

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. © 2016 American Society of Plant Biologists. All Rights Reserved.

Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

PubMed

Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

2005-01-01

Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region

PubMed Central

Philippe, Lucas; Vasseur, Jean-Jacques; Debart, Françoise

2018-01-01

Abstract Cell growth is a complex process shaped by extensive and coordinated changes in gene expression. Among these is the tightly regulated translation of a family of growth-related mRNAs defined by a 5′ terminal oligopyrimidine (TOP) motif. TOP mRNA translation is partly controlled via the eukaryotic initiation factor 4F (eIF4F), a translation factor that recognizes the mRNA 5′ cap structure. Recent studies have also implicated La-related protein 1 (LARP1), which competes with eIF4F for binding to mRNA 5′ ends. However, it has remained controversial whether LARP1 represses TOP mRNA translation directly and, if so, what features define its mRNA targets. Here, we show that the C-terminal half of LARP1 is necessary and sufficient to control TOP mRNA translation in cells. This fragment contains the DM15 cap-binding domain as well as an adjacent regulatory region that we identified. We further demonstrate that purified LARP1 represses TOP mRNA translation in vitro through the combined recognition of both the TOP sequence and cap structure, and that its intrinsic repressive activity and affinity for these features are subject to regulation. These results support a model whereby the translation of TOP mRNAs is controlled by a growth-regulated competition between eIF4F and LARP1 for their 5′ ends. PMID:29244122

Characteristics of the ToxRefDB In Vivo Datasets from Chronic, Reproductive and Developmental Assays

EPA Science Inventory

ToxRefDB was developed to store data from in vivo animal toxicity studies. The initial focus was populating ToxRefDB with pesticide registration toxicity data that has been historically stored as hard-copy and scanned documents by the Office of Pesticide Programs. A significant p...

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Yuen; Wang, Yuan; Feng, Jinyan

2015-04-03

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within itsmore » 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.« less

mRNA export in the apicomplexan parasite Toxoplasma gondii: emerging divergent components of a crucial pathway.

PubMed

Ávila, Andréa Rodrigues; Cabezas-Cruz, Alexjandro; Gissot, Mathieu

2018-01-25

Control of gene expression is crucial for parasite survival and is the result of a series of processes that are regulated to permit fine-tuning of gene expression in response to biological changes during the life-cycle of apicomplexan parasites. Control of mRNA nuclear export is a key process in eukaryotic cells but is poorly understood in apicomplexan parasites. Here, we review recent knowledge regarding this process with an emphasis on T. gondii. We describe the presence of divergent orthologs and discuss structural and functional differences in export factors between apicomplexans and other eukaryotic lineages. Undoubtedly, the use of the CRISPR/Cas9 system in high throughput screenings associated with the discovery of mRNA nuclear export complexes by proteomic analysis will contribute to identify these divergent factors. Ligand-based or structure-based strategies may be applied to investigate the potential use of these proteins as targets for new antiprotozoal agents.

Expression of phosphatidylserine-specific phospholipase A(1) mRNA in human THP-1-derived macrophages.

PubMed

Hosono, Hiroyuki; Homma, Masato; Ogasawara, Yoko; Makide, Kumiko; Aoki, Junken; Niwata, Hideaki; Watanabe, Machiko; Inoue, Keizo; Ohkohchi, Nobuhiro; Kohda, Yukinao

2010-01-01

The expression of phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) is most upregulated in the genes of peripheral blood cells from chronic rejection model rats bearing long-term surviving cardiac allografts. The expression profile of PS-PLA(1) in peripheral blood cells responsible for the immune response may indicate a possible biological marker for rejection episodes. In this study, PS-PLA(1) mRNA expression was examined in human THP-1-derived macrophages. The effects of several immunosuppressive agents on this expression were also examined in in vitro experiments. A real-time RT-PCR analysis revealed that PS-PLA(1) mRNA expression was found in human THP-1-derived macrophages. This expression was enhanced in the cells stimulated with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. Other TLR ligands (TLR2, 3, 5, 7, and 9) did not show a significant induction of PS-PLA(1) mRNA. The time course of the mRNA expression profiles was different between PS-PLA(1) and tumor necrosis factor-α (TNF-α), which showed a maximal expression at 12 and 1 h after LPS stimulation, respectively. Among the observed immunosuppressive agents, corticosteroids, prednisolone, 6α-methylprednisolone, dexamethasone, and beclomethasone inhibited PS-PLA(1) expression with half-maximal inhibitory concentrations less than 3.0 nM, while methotrexate, cyclosporine A, tacrolimus, 6-mercaptopurine, and mycophenoic acid showed either a weak or moderate inhibition. These results suggest that the expression of PS-PLA(1) mRNA in THP-1-derived macrophages is activated via TLR4 and it is inhibited by corticosteroids, which are used at high dosages to suppress chronic allograft rejection.

KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

PubMed Central

Bird, Clark W.; Gardiner, Amy S.; Bolognani, Federico; Tanner, Daniel C.; Chen, Ching-Yi; Lin, Wei-Jye; Yoo, Soonmoon; Twiss, Jeffery L.; Perrone- Bizzozero, Nora

2013-01-01

The KH-type splicing regulatory protein (KSRP) promotes the decay of AU-rich element (ARE)-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4). Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43′s ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp+/− and Ksrp −/−embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3′ UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs. PMID:24244461

Selective probing of mRNA expression levels within a living cell.

PubMed

Nawarathna, D; Turan, T; Wickramasinghe, H Kumar

2009-08-24

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high ( approximately 2500) and extremely low (11 0) copy number mRNA from a living cell mRNA in less than 10 s.

Selective probing of mRNA expression levels within a living cell

PubMed Central

Nawarathna, D.; Turan, T.; Wickramasinghe, H. Kumar

2009-01-01

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high (∼2500) and extremely low (11¯0) copy number mRNA from a living cell mRNA in less than 10 s. PMID:19777090

mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

2008-12-01

Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

PubMed

Lyabin, D N; Ovchinnikov, L P

2016-03-02

The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

Differential effects of voluntary wheel running and toy rotation on the mRNA expression of neurotrophic factors and FKBP5 in a post-traumatic stress disorder rat model with the shuttle-box task.

PubMed

Tanichi, Masaaki; Toda, Hiroyuki; Shimizu, Kunio; Koga, Minori; Saito, Taku; Enomoto, Shingo; Boku, Shuken; Asai, Fumiho; Mitsui, Yumi; Nagamine, Masanori; Fujita, Masanori; Yoshino, Aihide

2018-06-18

Life-threatening experiences can result in the development of post-traumatic stress disorder. We have developed an animal model for post-traumatic stress disorder (PTSD) using a shuttle box in rats. In this paradigm, the rats were exposed to inescapable foot-shock stress (IS) in a shuttle box, and then an avoidance/escape task was performed in the same box 2 weeks after IS. A previous study using this paradigm revealed that environmental enrichment (EE) ameliorated avoidance/numbing-like behaviors, but not hyperarousal-like behaviors, and EE also elevated hippocampal brain-derived neurotrophic factor (BDNF) expression. However, the differential effects of EE components, i.e., running wheel (RW) or toy rotation, on PTSD-like behaviors has remained unclear. In this experiment, we demonstrated that RW, toy rotation, and EE (containing RW and toy rotation) ameliorated avoidance/numbing-like behaviors, induced learning of avoidance responses, and improved depressive-like behaviors in traumatized rats. The RW increased the hippocampal mRNA expression of neurotrophic factors, especially BDNF and glial-cell derived neurotrophic factor. Toy rotation influenced FK506 binding protein 5 mRNA expression, which is believed to be a regulator of the hypothalamic-pituitary-adrenal (HPA)-axis system, in the hippocampus and amygdala. This is the first report to elucidate the differential mechanistic effects of RW and toy rotation. The former appears to exert its effects via neurotrophic factors, while the latter exerts its effects via the HPA axis. Further studies will lead to a better understanding of the influence of environmental factors on PTSD. Copyright © 2018 Elsevier Inc. All rights reserved.

Altered PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression in ejaculated spermatozoa of men with impaired sperm characteristics.

PubMed

Giebler, Maria; Greither, Thomas; Müller, Lisa; Mösinger, Carina; Behre, Hermann M

2018-01-01

In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1-4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5 th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1-4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1-4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1-4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1-4 mRNA expression on male infertility.

In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis.

PubMed

Feliciani, C; Toto, P; Amerio, P; Pour, S M; Coscione, G; Shivji, G; Wang, B; Sauder, D N

2000-01-01

Keratinocyte-derived cytokines have been implicated in the pathogenesis of a number of skin diseases. In this study we examined the possible role of keratinocyte-derived cytokines in the development of acantholysis in pemphigus vulgaris. Nineteen patients with pemphigus vulgaris, demonstrating the characteristic clinical, pathologic, and immunopathologic findings were studied. In situ immunolabeling demonstrated the presence of two cytokines interleukin-1alpha and tumor necrosis factor-alpha, in lesional and perilesional areas. Results were confirmed by reverse transcriptase-polymerase chain reaction, demonstrating overexpression of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of pemphigus vulgaris both in vitro and in vivo studies were performed. The results of the in vitro study demonstrated that pemphigus vulgaris IgG induced interleukin-1alpha and tumor necrosis factor-alpha mRNA in the skin. The potential pathogenic role of these mediators was demonstrated by a blocking study using antibodies against human interleukin-1alpha and tumor necrosis factor-alpha in keratinocytes cultures. A combination of anti-interleukin-1alpha and anti-tumor necrosis factor-alpha antibodies inhibited in vitro pemphigus vulgaris IgG induced acantholysis. To confirm the role of interleukin-1 and tumor necrosis factor-alpha in pemphigus, we utilized passive transfer studies using interleukin-1 deficient mice (ICE-/-, interleukin-1beta-/-) and tumor necrosis factor-alpha receptor deficient mice (TNFR1R2-/-). Both groups demonstrated a decreased susceptibility to the passive transfer of pemphigus. Our data support the role of cytokines interleukin-1 and tumor necrosis factor-alpha in the pathogenesis of pemphigus vulgaris.

HuR (Elavl1) and HuB (Elavl2) Stabilize Matrix Metalloproteinase-9 mRNA During Seizure-Induced Mmp-9 Expression in Neurons

PubMed Central

Zybura-Broda, Katarzyna; Wolder-Gontarek, Malgorzata; Ambrozek-Latecka, Magdalena; Choros, Artur; Bogusz, Agnieszka; Wilemska-Dziaduszycka, Joanna; Rylski, Marcin

2018-01-01

Matrix metalloproteinase-9 (Mmp-9) is involved in different general and cell-type–specific processes, both in neuronal and non-neuronal cells. Moreover, it is implicated in an induction or progression of various human disorders, including diseases of the central nervous system. Mechanisms regulating activity-driven Mmp-9 expression in neurons are still not fully understood. Here, we show that stabilization of Mmp-9 mRNA is one of the factors responsible for the neuronal activity-evoked upregulation of Mmp-9 mRNA expression in hippocampal neurons. Furthermore, we demonstrate that the molecular mechanism related to this stabilization is dependent on the neuronal seizure-triggered transiently increased binding of the mRNA stability-inducing protein, HuR, to ARE1 and ARE4 motifs of the 3′UTR for Mmp-9 mRNA as well as the stably augmented association of another mRNA-stabilizing protein, HuB, to the ARE1 element of the 3′UTR. Intriguingly, we demonstrate further that both HuR and HuB are crucial for an incidence of Mmp-9 mRNA stabilization after neuronal activation. This study identifies Mmp-9 mRNA as the first HuB target regulated by mRNA stabilization in neurons. Moreover, these results are the first to describe an existence of HuR-dependent mRNA stabilization in neurons of the brain. PMID:29686606

NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway

PubMed Central

Bühlmann, Melanie; Walrad, Pegine; Rico, Eva; Ivens, Alasdair; Capewell, Paul; Naguleswaran, Arunasalam; Roditi, Isabel; Matthews, Keith R.

2015-01-01

Trypanosomes mostly regulate gene expression through post-transcriptional mechanisms, particularly mRNA stability. However, much mRNA degradation is cytoplasmic such that mRNA nuclear export must represent an important level of regulation. Ribosomal RNAs must also be exported from the nucleus and the trypanosome orthologue of NMD3 has been confirmed to be involved in rRNA processing and export, matching its function in other organisms. Surprisingly, we found that TbNMD3 depletion also generates mRNA accumulation of procyclin-associated genes (PAGs), these being co-transcribed by RNA polymerase I with the procyclin surface antigen genes expressed on trypanosome insect forms. By whole transcriptome RNA-seq analysis of TbNMD3-depleted cells we confirm the regulation of the PAG transcripts by TbNMD3 and using reporter constructs reveal that PAG1 regulation is mediated by its 5′UTR. Dissection of the mechanism of regulation demonstrates that it is not dependent upon translational inhibition mediated by TbNMD3 depletion nor enhanced transcription. However, depletion of the nuclear export factors XPO1 or MEX67 recapitulates the effects of TbNMD3 depletion on PAG mRNAs and mRNAs accumulated in the nucleus of TbNMD3-depleted cells. These results invoke a novel RNA regulatory mechanism involving the NMD3-dependent nuclear export of mRNA cargos, suggesting a shared platform for mRNA and rRNA export. PMID:25873624

Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

PubMed

Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

2017-05-04

Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Stability of the Osmoregulated Promoter-Derived proP mRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli

PubMed Central

Lim, Boram

2015-01-01

ABSTRACT The enzymatic activity of Escherichia coli endo-RNase III determines the stability of a subgroup of mRNA species, including bdm, betT, and proU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability of proP mRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels of proP mRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell. In vitro and in vivo analyses of proP mRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only in proP mRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III on proP mRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance of proP mRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III via in vivo cross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress in E. coli. IMPORTANCE Our results demonstrate that RNase III activity on proP mRNA degradation is downregulated in Escherichia coli cells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of

Conjunctival mucin mRNA expression in contact lens wear.

PubMed

Corrales, Rosa M; Galarreta, David; Herreras, Jose M; Saez, Victoria; Arranz, Isabel; González, Maria J; Mayo, Agustin; Calonge, Margarita; Chaves, Felipe J

2009-09-01

To investigate the influence of the water content in non-ionic hydrogel contact lenses (HCL) on the mRNA levels of human conjunctival mucin genes (MUCs). Sixteen healthy subjects with no history of contact lenses wear were selected and randomized into two equal groups. Group 1 subjects wore low water content (38%, Soflens 38) non-ionic HCLs. Group 2 wore high water content (66%, Soflens 66) non-ionic HCLs. Conjunctival impression cytology was applied to the superior bulbar conjunctiva of both eyes before, 6 months, and 1 year after HCL fitting, and 15 days after discontinuation of wearing. Total RNA was isolated, retrotranscribed, and amplified by conventional polymerase chain reaction (PCR) and by quantitative real time PCR to study the mRNA levels of MUCs and to analyze variations during the study period. Time- and HCL-dependent variations in mRNA expression were analyzed using Student's test. From the known MUCs, transcripts from MUC1, MUC2, MUC4, MUC5AC, MUC7, MUC13, MUC15, MUC16, and MUC17 genes were detected in all subjects before HCL fitting. Except for MUC2, the expression of some MUC genes significantly increased whereas others significantly decreased at either the 6- and 12-month period. Statistically significant differences between both HCL groups (p < 0.001) were found in the MUC4, MUC13, and MUC15 mRNA expression after 1 year of wear and after the 15 days without HCL wear. However, these differences were not clearly related to the water content of the lenses. Low and high water content non-ionic HCLs induced different changes in the mRNA levels of several MUCs, but the water content was not related to the changes. Recovery to basal levels of conjunctival MUC mRNA expression after wearing HCL lenses for a year takes longer than 15 days for some MUCs.

c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells.

PubMed

Biskobing, D M; Fan, D; Rubin, J

1997-09-01

Macrophage colony-stimulating factor (MCSF) is required for normal osteoclast and macrophage development. The receptor for MCSF (c-fms) is expressed on the pluripotent precursor and mature osteoclasts and macrophages. We have previously shown in myelomonocytic HL-60 cells that phorbol myristate acetate (PMA) upregulates c-fms mRNA expression. This induction of c-fms is inhibited by 1,25(OH)2D3. The major regulatory control of c-fms mRNA levels by PMA has been identified as posttranscriptional. However, a role of transcript elongation in controlling levels of c-fms mRNA has also been suggested. To better understand the 1,25(OH)2D3 regulation of c-fms mRNA expression we studied nuclear run on, mRNA stability, and transcript elongation in HL-60 cells treated with 10 ng/ml phorbol myristate acetate, 10 nM 1,25(OH)2D3 alone or combined. We demonstrated by nuclear run on that c-fms was constitutively transcribed in 1,25(OH)2D3 as well as control and PMA-treated cells. Transcript elongation was evaluated by RT-PCR for exon 2 or exon 3. Both exons were minimally expressed in control and 1,25(OH)2D3-treated cells, and increased in PMA-treated cells; this increased expression was inhibited by the addition of 1,25(OH)2D3. These results fail to show differential transcript elongation. Measurement of mRNA stability demonstrated decreased mRNA half-life to 5 hours in cells treated with PMA and 1,25(OH)2D3 compared with a half-life of 8 hours in cells treated with PMA alone. Our findings demonstrate that c-fms is regulated by 1,25(OH)2D3 at the posttranscriptional level by changes in mRNA stability. This gives the cell the ability to respond to local signals with rapid changes in c-fms levels altering the ability of the cell to respond to MCSF.

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

PubMed

Higashimoto, Yuji; Elliott, W Mark; Behzad, Ali R; Sedgwick, Edward G; Takei, Tatsuo; Hogg, James C; Hayashi, Shizu

2002-07-15

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

Regulation of mRNA Trafficking by Nuclear Pore Complexes

PubMed Central

Bonnet, Amandine; Palancade, Benoit

2014-01-01

Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662

WhiteRef: a new tower-based hyperspectral system for continuous reflectance measurements.

PubMed

Sakowska, Karolina; Gianelle, Damiano; Zaldei, Alessandro; MacArthur, Alasdair; Carotenuto, Federico; Miglietta, Franco; Zampedri, Roberto; Cavagna, Mauro; Vescovo, Loris

2015-01-08

Proximal sensing is fundamental to monitor the spatial and seasonal dynamics of ecosystems and can be considered as a crucial validation tool to upscale in situ observations to the satellite level. Linking hyperspectral remote sensing with carbon fluxes and biophysical parameters is critical to allow the exploitation of spatial and temporal extensive information for validating model simulations at different scales. In this study, we present the WhiteRef, a new hyperspectral system designed as a direct result of the needs identified during the EUROSPEC ES0903 Cost Action, and developed by Fondazione Edmund Mach and the Institute of Biometeorology, CNR, Italy. The system is based on the ASD FieldSpec Pro spectroradiometer and was designed to acquire continuous radiometric measurements at the Eddy Covariance (EC) towers and to fill a gap in the scientific community: in fact, no system for continuous spectral measurements in the Short Wave Infrared was tested before at the EC sites. The paper illustrates the functioning of the WhiteRef and describes its main advantages and disadvantages. The WhiteRef system, being based on a robust and high quality commercially available instrument, has a clear potential for unattended continuous measurements aiming at the validation of satellites' vegetation products.

Test Review: Delis, D. (2012) "Delis Rating of Executive Function (D-REF)." Bloomington, Minnesota: Pearson

ERIC Educational Resources Information Center

Warnick, Brittany; Drake, Morgan; Vidrine, Stephanie

2015-01-01

The Delis Rating of Executive Function (D-REF) is a set of rating scales designed to assess executive functions and their constituent sub-processes in children and adolescents between the ages of 5 and 18. More specifically, the D-REF is a supplemental assessment of children and adolescents demonstrating behavioral or cognitive difficulties often…

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

PubMed

Kanai, Yukiko; Asano, Kosuke; Komatsu, Yoshiko; Takagi, Katsuhiro; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Haneishi, Ayumi; Tsukada, Akiko; Yamada, Kazuya

2017-02-01

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

Impact of STAT/SOCS mRNA Expression Levels after Major Injury

PubMed Central

Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

2014-01-01

Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

Generation and Evaluation of Prophylactic mRNA Vaccines Against Allergy.

PubMed

Weiss, Richard; Scheiblhofer, Sandra; Thalhamer, Josef

2017-01-01

Due to the worldwide increase in allergies and a limited efficacy of therapeutic interventions, the need for prophylactic vaccination against allergies has been recognized. mRNA and DNA vaccines have demonstrated their high potential for preventing allergic sensitization by inducing an immunological bias that prevents TH2 sensitization. However, only mRNA vaccines fulfill the stringent safety requirements for vaccination of healthy children. In this chapter, we describe the generation of conventional as well as self-replicating mRNA vaccines and methods to test their prophylactic efficacy in animal models.

Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

PubMed Central

Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

2013-01-01

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

PubMed Central

Nie, Mei; Balda, Maria S.; Matter, Karl

2012-01-01

A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O.

1988-11-01

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA andmore » DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.« less

[Protein S3 fragments neighboring mRNA during elongation and translation termination on the human ribosome].

PubMed

Khaĭrulina, Iu S; Molotkov, M V; Bulygin, K N; Graĭfer, D M; Ven'yaminova, A G; Frolova, L Iu; Stahl, J; Karpova, G G

2008-01-01

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site binding codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5'-termini that could predetermine the position of the tRNA(Phe) on the ribosome by the location of P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3' of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide-induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2-127) and/or in the C-terminal fragment 190-236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.

Role of mRNA Methylation in Prostate Cancer

DTIC Science & Technology

2015-02-01

position of adenosine (m6A) is a post-transcriptional modification of mRNA. However, little is known regarding the biological meanings of this epigenetic ...its level is altered in various cancer cell lines. FTO, the fat mass and obesity associated gene, was recently shown as an m6A demethylase. FTO gene...mRNA. However, little is known regarding the biological meanings of this epigenetic regulation of mRNA. Recent technological advances have made it

Preprotachykinin A mRNA expression in the rat brain during development.

PubMed

Brené, S; Lindefors, N; Friedman, W J; Persson, H

1990-12-15

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.

Architecture of eukaryotic mRNA 3′-end processing machinery

PubMed Central

Hill, Chris H.; Easter, Ashley D.; Emsley, Paul; Degliesposti, Gianluca; Gordiyenko, Yuliya; Santhanam, Balaji; Wolf, Jana; Wiederhold, Katrin; Dornan, Gillian L.; Skehel, Mark; Robinson, Carol V.; Passmore, Lori A.

2018-01-01

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing. PMID:29074584

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

PubMed

Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

1997-07-01

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Polymorphism at the ref(2)P locus in Drosophila melanogaster: preliminary experiments concerning the selection mechanisms involved in its maintenance.

PubMed

Fleuriet, A

1981-02-01

It has been shown previously that a polymorphism for two alleles of the ref(2)P locus is a regular feature of French natural populations of Drosophila melanogaster and that this is maintained in laboratory populations raised in cages. In this paper, an experimental population and egg-collection experiments are reported. Differential survival of the three genotypes would be the main factor leading to the equilibrium frequencies, working only in drastic conditions of larval competition.

Structural model of an mRNA in complex with the bacterial chaperone Hfq

DOE PAGES

Peng, Yi; Curtis, Joseph E.; Fang, Xianyang; ...

2014-11-17

The Sm-like protein Hfq (host factor Q-beta phage) facilitates regulation by bacterial small noncoding RNAs (sRNAs) in response to stress and other environmental signals. In this paper, we present a low-resolution model of Escherichia coli Hfq bound to the rpoS mRNA, a bacterial stress response gene that is targeted by three different sRNAs. Selective 2'-hydroxyl acylation and primer extension, small-angle X-ray scattering, and Monte Carlo molecular dynamics simulations show that the distal face and lateral rim of Hfq interact with three sites in the rpoS leader, folding the RNA into a compact tertiary structure. These interactions are needed for sRNAmore » regulation of rpoS translation and position the sRNA target adjacent to an sRNA binding region on the proximal face of Hfq. Finally, our results show how Hfq specifically distorts the structure of the rpoS mRNA to enable sRNA base pairing and translational control.« less

Delta ribozyme has the ability to cleave in transan mRNA.

PubMed Central

Roy, G; Ananvoranich, S; Perreault, J P

1999-01-01

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724

Green tea polyphenols improve cardiac muscle mRNA and protein levels of signal pathways related to insulin and lipid metabolism and inflammation in insulin-resistant rats.

PubMed

Qin, Bolin; Polansky, Marilyn M; Harry, Dawson; Anderson, Richard A

2010-05-01

Epidemiological studies indicate that the consumption of green tea polyphenols (GTP) may reduce the risk of coronary artery disease. To explore the underlying mechanisms of action at the molecular level, we examined the effects of GTP on the cardiac mRNA and protein levels of genes involved in insulin and lipid metabolism and inflammation. In rats fed a high-fructose diet, supplementation with GTP (200 mg/kg BW daily dissolved in distilled water) for 6 wk, reduced systemic blood glucose, plasma insulin, retinol-binding protein 4, soluble CD36, cholesterol, triglycerides, free fatty acids and LDL-C levels, as well as the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and IL-6. GTP did not affect food intake, bodyweight and heart weight. In the myocardium, GTP also increased the insulin receptor (Ir), insulin receptor substrate 1 and 2 (Irs1 and Irs2), phosphoinositide-3-kinase (Pi3k), v-akt murine thymoma viral oncogene homolog 1 (Akt1), glucose transporter 1 and 4 (Glut1 and Glut4) and glycogen synthase 1 (Gys1) expression but inhibited phosphatase and tensin homolog deleted on chromosome ten (Pten) expression and decreased glycogen synthase kinase 3beta (Gsk3beta) mRNA expression. The sterol regulatory element-binding protein-1c (Srebp1c) mRNA, microsomal triglyceride transfer protein (Mttp) mRNA and protein, Cd36 mRNA and cluster of differentiation 36 protein levels were decreased and peroxisome proliferator-activated receptor (Ppar)gamma mRNA levels were increased. GTP also decreased the inflammatory factors: Tnf, Il1b and Il6 mRNA levels, and enhanced the anti-inflammatory protein, zinc-finger protein, protein and mRNA expression. In summary, consumption of GTP ameliorated the detrimental effects of high-fructose diet on insulin signaling, lipid metabolism and inflammation in the cardiac muscle of rats.

RNActive® Technology: Generation and Testing of Stable and Immunogenic mRNA Vaccines.

PubMed

Rauch, Susanne; Lutz, Johannes; Kowalczyk, Aleksandra; Schlake, Thomas; Heidenreich, Regina

2017-01-01

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon

2012-03-16

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosenmore » DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.« less

Lymphotoxin β receptor activation promotes mRNA expression of RelA and pro-inflammatory cytokines TNFα and IL-1β in bladder cancer cells.

PubMed

Shen, Mo; Zhou, Lianlian; Zhou, Ping; Zhou, Wu; Lin, Xiangyang

2017-07-01

The role of inflammation in tumorigenesis and development is currently well established. Lymphotoxin β receptor (LTβR) activation induces canonical and noncanonical nuclear factor (NF)‑κB signaling pathways, which are linked to inflammation‑induced carcinogenesis. In the present study, 5,637 bladder cancer cells were cultured and the activation of LTβR was induced by functional ligand, lymphotoxin (LT) α1β2, and silencing with shRNA. Reverse transcription‑quantitative polymerase chain reaction was utilized to detect the mRNA expression levels of NF‑κB family members RelA and RelB, cytokines including LTα, LTβ, tumor necrosis factor (TNF)α, TNF superfamily member 14, interleukin (IL)‑6 and IL‑1β, and proliferation‑related genes including CyclinD1 and Survivin. The expression of phospho‑p65 was determined by western blotting. Activation of LTβR on bladder cancer 5,637 cells was demonstrated to upregulate the mRNA expression levels of the RELA proto‑oncogene, RelA, by 2.5‑fold compared with unstimulated cells, while no significant change was observed in the RELB proto‑oncogene NF‑κB member mRNA levels. Expression of pro‑inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)‑1β mRNA levels were significantly increased nearly 5‑fold and 1.5‑fold, respectively, following LTβR activation compared with unstimulated cells. The LTβR‑induced upregulation of RelA, TNFα and IL‑1β was decreased by ~33, 27, and 26% respectively when LTβR was silenced via short hairpin RNA. Activation of LTβR had no effect on 5,637 cell growth, despite CyclinD1 and Survivin mRNA levels increasing by ~2.7 and 1.3‑fold, respectively, compared with unstimulated cells. In conclusion, activation of LTβR induced the expression of RelA mRNA levels. LTβR activation might be an important mediator in promoting an inflammatory microenvironment in bladder cancer, via the upregulation of TNFα and IL‑1β mRNA levels. LTβR may

Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

PubMed

Soetanto, R; Hynes, C J; Patel, H R; Humphreys, D T; Evers, M; Duan, G; Parker, B J; Archer, S K; Clancy, J L; Graham, R M; Beilharz, T H; Smith, N J; Preiss, T

2016-05-01

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. Copyright © 2016 Elsevier B.V. All rights reserved.

Cis-Regulatory Variants Affect CHRNA5 mRNA Expression in Populations of African and European Ancestry

PubMed Central

Wang, Jen-Chyong; Spiegel, Noah; Bertelsen, Sarah; Le, Nhung; McKenna, Nicholas; Budde, John P.; Harari, Oscar; Kapoor, Manav; Brooks, Andrew; Hancock, Dana; Tischfield, Jay; Foroud, Tatiana; Bierut, Laura J.; Steinbach, Joe Henry; Edenberg, Howard J.; Traynor, Bryan J.; Goate, Alison M.

2013-01-01

Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels. PMID:24303001

Assessing mRNA nuclear export in mammalian cells by microinjection.

PubMed

Lee, Eliza S; Palazzo, Alexander F

2017-08-15

The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis

PubMed Central

Natsuizaka, Mitsuteru; Naganuma, Seiji; Kagawa, Shingo; Ohashi, Shinya; Ahmadi, Azal; Subramanian, Harry; Chang, Sanders; Nakagawa, Kei J.; Ji, Xinjun; Liebhaber, Stephen A.; Klein-Szanto, Andres J.; Nakagawa, Hiroshi

2012-01-01

Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein-Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis. PMID:22415309

Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

PubMed

Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

2004-03-17

Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

RefEx, a reference gene expression dataset as a web tool for the functional analysis of genes.

PubMed

Ono, Hiromasa; Ogasawara, Osamu; Okubo, Kosaku; Bono, Hidemasa

2017-08-29

Gene expression data are exponentially accumulating; thus, the functional annotation of such sequence data from metadata is urgently required. However, life scientists have difficulty utilizing the available data due to its sheer magnitude and complicated access. We have developed a web tool for browsing reference gene expression pattern of mammalian tissues and cell lines measured using different methods, which should facilitate the reuse of the precious data archived in several public databases. The web tool is called Reference Expression dataset (RefEx), and RefEx allows users to search by the gene name, various types of IDs, chromosomal regions in genetic maps, gene family based on InterPro, gene expression patterns, or biological categories based on Gene Ontology. RefEx also provides information about genes with tissue-specific expression, and the relative gene expression values are shown as choropleth maps on 3D human body images from BodyParts3D. Combined with the newly incorporated Functional Annotation of Mammals (FANTOM) dataset, RefEx provides insight regarding the functional interpretation of unfamiliar genes. RefEx is publicly available at http://refex.dbcls.jp/.

An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export

PubMed Central

Jackson, Brian R.; Boyne, James R.; Noerenberg, Marko; Taylor, Adam; Hautbergue, Guillaume M.; Walsh, Matthew J.; Wheat, Rachel; Blackbourn, David J.; Wilson, Stuart A.; Whitehouse, Adrian

2011-01-01

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs. PMID:21814512

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

PubMed

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

Single step production of Cas9 mRNA for zygote injection.

PubMed

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Magnesium Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

PubMed Central

Swift, Robert V.; Ong, Chau D.; Amaro, Rommie E.

2012-01-01

The messenger RNA guanylyltransferase, or mRNA capping enzyme, co-transcriptionally caps the 5′-end of nascent mRNA with GMP during the second in a set of three enzymatic reactions that result in the formation of an N7-methyl guanosine cap during mRNA maturation. The mRNA capping enzyme is characterized, in part, by a conserved lysine nucleophile that attacks the alpha-phosphorous atom of GTP, forming a lysine-GMP intermediate. Experiments have firmly established that magnesium is required for efficient intermediate formation, but have provided little insight into the requirement’s molecular origins. Using empirical and thermodynamic integration pKa estimates, along with conventional MD simulations, we show that magnesium binding likely activates the lysine nucleophile by increasing its acidity and by biasing the deprotonated nucleophile into conformations conducive to intermediate formation. These results provide additional functional understanding of an important enzyme in the mRNA transcript life cycle and allow functional analogies to be drawn that affect our understanding of the metal dependence of related superfamily members. PMID:23205906

Polymorphism at the REF(2)P Locus in DROSOPHILA MELANOGASTER: Preliminary Experiments concerning the Selection Mechanisms Involved in Its Maintenance

PubMed Central

Fleuriet, Annie

1981-01-01

It has been shown previously that a polymorphism for two alleles of the ref(2)P locus is a regular feature of French natural populations of Drosophila melanogaster and that this is maintained in laboratory populations raised in cages. In this paper, an experimental population and egg-collection experiments are reported. Differential survival of the three genotypes would be the main factor leading to the equilibrium frequencies, working only in drastic conditions of larval competition. PMID:6791986

Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

PubMed Central

Popp, Maximilian Wei-Lin; Maquat, Lynne E.

2014-01-01

Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control. PMID:24274751

Heparanase mRNA expression and point mutation in hepatocellular carcinoma

PubMed Central

Chen, Xiao-Peng; Liu, Yin-Bib; Rui, Jing; Peng, Shu-You; Peng, Cheng-Hong; Zhou, Zi-Yan; Shi, Liang-Hui; Shen, Hong-Wei; Xu, Bin

2004-01-01

AIM: To explore the expression of heparanase mRNA and point mutation in hepatocellular carcinoma (HCC). METHODS: Reverse transcription polymerase chain reaction was used to measure the expression of heparanase mRNA in the primary tumor tissues and surrounding liver tissues of 33 HCC patients. T-A cloning and sequencing were used to detect whether there was any mutation in the amplified PCR products. RESULTS: The expression of heparanase mRNA was positive in 16 primary tumor tissues of HCC, and the positive rate was 48.5%, which was significantly higher than that in the surrounding liver parenchyma (P < 0.01). The positive rate for heparanase gene in high-tendency to metastatic recurrence group (71.4%, 10/14) was obviously higher than that in low-tendency to metastatic recurrence group (31.6%, 6/19) (P = 0.023). The positive rate for heparanase gene in patients with metastatic recurrence during postoperative follow-up (78.6%, 11/14) was also significantly higher than that in those without metastatic recurrence (21.4%, 3/14) (P = 0.003). Sequence analysis of the HPA PCR products was made in 7 patients, and 2-point mutations were found in 4 patients, one of which was sense mutation, neither base insertion nor deletion was detected. The mutation rate was 57.1% (4/7). CONCLUSION: The expression rate of heparanase mRNA increases in HCC, and HPA mRNA may be one of the reliable markers for the metastatic activity gained by the liver tumor cells and could be used clinically in predicting metastatic recurrence of HCC. Point mutation may be one of the causes for enhanced heparanase mRNA expression. PMID:15334672

Diagnostic performance of HPV E6/E7 mRNA assay for detection of cervical high-grade intraepithelial neoplasia and cancer among women with ASCUS Papanicolaou smears.

PubMed

Ren, Chenchen; Zhu, Yuanhang; Yang, Li; Zhang, Xiaoan; Liu, Ling; Ren, Chunying

2018-02-01

The aim of this study was to investigate the clinical performance of high risk (HR) HPV E6/E7 mRNA assay in detecting cervical high-grade intraepithelial neoplasia and cancer among women with atypical squamous cells of undetermined significance (ASCUS) Papanicolaou (Pap) smears. A total of 160 patients with ASCUS who underwent HR-HPV DNA assay, HR-HPV E6/E7 mRNA assay and colposcopy biopsy at Third Affiliated Hospital of Zhengzhou University, China, from December 2015 to March 2017, were enrolled. Logistic regression analysis was used to evaluate the relationship between pathological results with clinical biologic factors. Univariate analysis showed that the qualitative results of HR-HPV DNA, qualitative results of HR-HPV E6/E7 mRNA and expression levels of HR-HPV E6/E7 mRNA were risk factors of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (all P < 0.05). Multivariable analysis found that only the expression levels of HR-HPV E6/E7 mRNA was associated with high-grade CIN and cervical cancer (OR = 8.971, 95% CI = 2.572-31.289, P = 0.001). An optimal cut-off value of ≥ 558.26 copies/ml was determined using receiver operating characteristic curve, and specificity of cut-off value were higher than E6/E7 mRNA qualitative assay and DNA qualitative assay. HPV E6/E7 mRNA quantitative assay may be a valuable tool in triage of ASCUS pap smears. A high specificity of E6/E7 mRNA quantitative assay as a triage test in women with ASCUS can be translated into a low referral for colposcopy.

Peripheral mRNA expression of pluripotency markers in bipolar disorder and the effect of long-term lithium treatment.

PubMed

Ferensztajn-Rochowiak, Ewa; Tarnowski, Maciej; Samochowiec, Jerzy; Michalak, Michal; Ratajczak, Mariusz Z; Rybakowski, Janusz K

2016-10-01

The aim was to evaluate the peripheral mRNA expression of pluripotency master transcriptional factors such as octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2) and homeobox protein Nanog, in patients with bipolar disorder (BD), and the effect of long-term lithium treatment. Fifteen BD patients (aged 53±7years) not treated with lithium, with duration of illness>10years, 15 BD patients (aged 55±6years) treated with lithium for 8-40 years (mean 16years) and 15 control subjects (aged 50±5years) were included. Assessment of the mRNA levels of pluripotency markers (Oct-4, Sox 2 and Nanog) was performed, using the Real-time quantitative reverse transcription PCR (RQ-PCR) procedure, and the number of CD34+ very small embryonic-like stem cells (VSELs) was measured by flow cytometric analysis. In those BD patients not treated with lithium the expression of all three pluripotency genes was significantly higher than that in the control subjects. Oct-4, Sox2 and Nanog also positively correlated with the number of CD34+ VSELs/[ul] in this group. In the lithium-treated patients the mRNA levels of Nanog were significantly higher than in the control individuals and correlated with the number and % of CD34+ VSELs. The overexpression of the pluripotency master transcriptional factors in patients with a long duration of BD not treated with lithium, may contribute to the pathogenesis of the illness and make them potential biological markers of BD. Long-term lithium treatment may attenuate these excessive regenerative processes, especially in relation to the transcription factors Oct-4 and Sox2. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

PubMed Central

Kreth, Simone; Thon, Niklas; Eigenbrod, Sabina; Lutz, Juergen; Ledderose, Carola; Egensperger, Rupert; Tonn, Joerg C.; Kretzschmar, Hans A.; Hinske, Ludwig C.; Kreth, Friedrich W.

2011-01-01

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT

Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue

PubMed Central

CAVAZZOLA, LUCIANE ROSTIROLA; CARVALHAL, GUSTAVO FRANCO; DEVES, CANDIDA; RENCK, DAIANA; ALMEIDA, RICARDO; SANTOS, DIóGENES SANTIAGO

2015-01-01

Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. P<0.05 was considered to indicate a statistically significant difference. The results revealed that PDEF, ETV4, UP-1 and TP were expressed in 85.7, 90.5, 95.2 and 100% of the prostate cancer samples, and in 90.3, 96.8, 90.3 and 96.8% of the benign samples, respectively. PDEF and ETV4 exhibited a significantly higher relative expression level in the tumor samples compared with their benign counterparts. The relative expression of TP and UP-1 did not differ significantly between benign and cancerous prostate tissues. The relative expression of TP was moderately and significantly correlated with the expression of ETV4 in the benign tissues. The relative expression of UP-1 was significantly lower in T3 compared with T1 and T2 cancers. These findings indicate that PDEF, ETV4, TP and UP-1 are typically expressed in benign and malignant prostatic

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells.

PubMed

Philipp, Katrin; Riedel, Frank; Germann, Günter; Hörmann, Karl; Sauerbier, Michael

2005-02-01

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Caffeine Bitterness is Related to Daily Caffeine Intake and Bitter Receptor mRNA Abundance in Human Taste Tissue

PubMed Central

Lipchock, Sarah V.; Spielman, Andrew I.; Mennella, Julie A.; Mansfield, Corrine J.; Hwang, Liang-Dar; Douglas, Jennifer E.; Reed, Danielle R.

2018-01-01

We investigated whether the abundance of bitter receptor mRNA expression from human taste papillae is related to an individual’s perceptual ratings of bitter intensity and habitual intake of bitter drinks. Ratings of the bitterness of caffeine and quinine and three other bitter stimuli (urea, propylthiouracil, and denatonium benzoate) were compared with relative taste papilla mRNA abundance of bitter receptors that respond to the corresponding bitter stimuli in cell-based assays (TAS2R4, TAS2R10, TAS2R38, TAS2R43, and TAS2R46). We calculated caffeine and quinine intake from a food frequency questionnaire. The bitterness of caffeine was related to the abundance of the combined mRNA expression of these known receptors, r = 0.47, p = .05, and self-reported daily caffeine intake, t(18) = 2.78, p = .012. The results of linear modeling indicated that 47% of the variance among subjects in the rating of caffeine bitterness was accounted for by these two factors (habitual caffeine intake and taste receptor mRNA abundance). We observed no such relationships for quinine but consumption of its primary dietary form (tonic water) was uncommon. Overall, diet and TAS2R gene expression in taste papillae are related to individual differences in caffeine perception. PMID:28118781

Texture Analysis of Poly-Adenylated mRNA Staining Following Global Brain Ischemia and Reperfusion

PubMed Central

Szymanski, Jeffrey J.; Jamison, Jill T.; DeGracia, Donald J.

2011-01-01

Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 hour of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 hour reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion. PMID:21477879

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

PubMed Central

Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

1978-01-01

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein

PubMed Central

2004-01-01

The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30–90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA. PMID:15496143

GLUT3 protein and mRNA in autopsy muscle specimens

NASA Technical Reports Server (NTRS)

Stuart, C. A.; Wen, G.; Jiang, J.

1999-01-01

GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

RefEx, a reference gene expression dataset as a web tool for the functional analysis of genes

PubMed Central

Ono, Hiromasa; Ogasawara, Osamu; Okubo, Kosaku; Bono, Hidemasa

2017-01-01

Gene expression data are exponentially accumulating; thus, the functional annotation of such sequence data from metadata is urgently required. However, life scientists have difficulty utilizing the available data due to its sheer magnitude and complicated access. We have developed a web tool for browsing reference gene expression pattern of mammalian tissues and cell lines measured using different methods, which should facilitate the reuse of the precious data archived in several public databases. The web tool is called Reference Expression dataset (RefEx), and RefEx allows users to search by the gene name, various types of IDs, chromosomal regions in genetic maps, gene family based on InterPro, gene expression patterns, or biological categories based on Gene Ontology. RefEx also provides information about genes with tissue-specific expression, and the relative gene expression values are shown as choropleth maps on 3D human body images from BodyParts3D. Combined with the newly incorporated Functional Annotation of Mammals (FANTOM) dataset, RefEx provides insight regarding the functional interpretation of unfamiliar genes. RefEx is publicly available at http://refex.dbcls.jp/. PMID:28850115

Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

PubMed

Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

2015-03-15

In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks. Copyright © 2015 Elsevier B.V. All rights reserved.

Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

PubMed

Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

2017-09-01

Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Tissue-specific mRNA expression profiling in grape berry tissues

PubMed Central

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells.

PubMed

Kim, H; You, S; Foster, L K; Farris, J; Foster, D N

2001-08-23

The steady-state levels of p53 mRNA were dramatically lower in immortal chicken embryo fibroblast (CEF) cell lines compared to primary CEF cells. In the presence of cycloheximide (CHX), the steady-state levels of p53 mRNA markedly increased in immortal CEF cell lines, similar to levels found in primary cells. The de novo synthetic rates of p53 mRNA were relatively similar in primary and immortal cells grown in the presence or absence of CHX. Destabilization of p53 mRNA was observed in the nuclei of immortal, but not primary, CEF cells. The half-life of p53 mRNA in primary cells was found to be a relatively long 23 h compared to only 3 h in immortal cells. The expression of transfected p53 cDNA was inhibited in immortal cells, but restored upon CHX treatment. The 5'-region of the p53 mRNA was shown to be involved in the rapid p53 mRNA destabilization in immortal cells by expression analysis of 5'- and 3'-deleted p53 cDNAs as well as fusion mRNA constructs of N-terminal p53 and N-terminal deleted LacZ genes. Together, it is suggestive that the downregulation of p53 mRNA in immortal CEF cells occurs through a post-transcriptional destabilizing mechanism.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

PubMed

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-05-18

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

Associations of ACE Gene Insertion/Deletion Polymorphism, ACE Activity, and ACE mRNA Expression with Hypertension in a Chinese Population

PubMed Central

He, Qingfang; Fan, Chunhong; Yu, Min; Wallar, Gina; Zhang, Zuo-Feng; Wang, Lixin; Zhang, Xinwei; Hu, Ruying

2013-01-01

Background The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D, rs4646994) polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored. Methods The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension. Results The results of the multivariate logistic regression analysis showed that the additive model (ID, DD versus II) of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls. Conclusion ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. PMID:24098401

Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.

PubMed

Yoshigai, Emi; Hara, Takafumi; Araki, Yoshiro; Tanaka, Yoshito; Oishi, Masaharu; Tokuhara, Katsuji; Kaibori, Masaki; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2013-04-01

Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3' untranslated region (3'UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3'UTR. Furthermore, single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3'UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2'-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

IGF-1R mRNA expression is increased in obese children.

PubMed

Ricco, Rafaela Cristina; Ricco, Rubens Garcia; Queluz, Mariangela Carletti; de Paula, Mariana Teresa Sarti; Atique, Patricia Volpon; Custódio, Rodrigo José; Tourinho Filho, Hugo; Del Roio Liberatori, Raphael; Martinelli, Carlos Eduardo

2018-04-01

Obese children are often taller than age-matched subjects. Reports on GH and IGF-I levels in obese individuals are controversial, with normal and reduced GH-IGF-I levels having been reported in this group of patients. Thus, the aim of this study was to analyse insulin-like growth factor type 1 receptor (IGF-IR) mRNA expression in obese children. Forty-seven pre-pubertal children were included in this study: 29 were obese and taller than their target height, and 18 were normal eutrophic controls. Fasting blood samples were collected for IGF-IR mRNA expression in isolated lymphocytes and serum IGF-I, ALS, IGFBP-3, and IGFBP-1 concentration analysis. Relative IGF-IR gene expression (2 -ΔΔCT ) was significantly (P=0.025) higher in obese children (median 1.87) than in controls (1.15). Fourteen of the 29 obese subjects showed 2 -ΔΔCT values greater than or equal to 2, while only 2 individuals in the control group showed values above 2 (P=0.01). Obese children showed significantly (P=0.01) higher IGF-I concentrations than the control group (237ng/ml and 144ng/ml, respectively). Among obese patients, 65.5% had IGF-I values above the 75 percentile of the control group (P=0.02). ALS concentration was significantly (P=0.04) higher in the obese group, while IGFBP-3 levels were similar in obese and control children. IGFBP-1 concentration was lower in obese children, while insulin levels and HOMA-IR index were higher than in controls. The higher IGF-IR mRNA expression observed in obese children, associated with the higher IGF-I and ALS and the lower IGFBP-1 levels, suggest that the higher stature observed in these children may be due to increased IGF-I bioactivity. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Effect of Repeated Electroacupuncture Analgesia on Neurotrophic and Cytokine Factors in Neuropathic Pain Rats

PubMed Central

Wang, Junying; Duanmu, Chenlin; Feng, Xiumei; Yan, Yaxia

2016-01-01

Chronic pain is a common disability influencing quality of life. Results of previous studies showed that acupuncture has a cumulative analgesic effect, but the relationship with spinal cytokines neurotrophic factors released by astrocytes remains unknown. The present study was designed to observe the effect of electroacupuncture (EA) treatment on spinal cytokines neurotrophic factors in chronic neuropathic pain rats. The chronic neuropathic pain was established by chronic constrictive injury (CCI). EA treatment was applied at Zusanli (ST36) and Yanglingquan (GB34) (both bilateral) once a day, for 30 min. IL-1β mRNA, TNF-α mRNA, and IL-1 mRNA were detected by quantitative real-time PCR, and the proteins of BDNF, NGF, and NT3/4 were detected by Western blot. The expression levels of cytokines such as IL-1β mRNA, TNF-α mRNA, IL-6 mRNA, and neurotrophic factors such as BDNF, NGF, and NT3/4 in the spinal cord were increased significantly after CCI. The astrocytes released more IL-1β and BDNF after CCI. Repeated EA treatment could suppress the elevated expression of IL-1β mRNA, TNFα mRNA, and BDNF, NGF, and NT3/4 but had no effect on IL-6 mRNA. It is suggested that cytokines and neurotrophic factors which may be closely associated with astrocytes participated in the process of EA relieving chronic pain. PMID:27800006

Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

PubMed

Marçais, Antoine; Tomkowiak, Martine; Walzer, Thierry; Coupet, Charles-Antoine; Ravel-Chapuis, Aymeric; Marvel, Jacqueline

2006-10-01

Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

Relationship between serum IGF-1 and skeletal muscle IGF-1 mRNA expression to phosphocreatine recovery after exercise in obese men with reduced GH.

PubMed

Hamarneh, Sulaiman R; Murphy, Caitlin A; Shih, Cynthia W; Frontera, Walter; Torriani, Martin; Irazoqui, Javier E; Makimura, Hideo

2015-02-01

GH and IGF-1 are believed to be physiological regulators of skeletal muscle mitochondria. The objective of this study was to examine the relationship between GH/IGF-1 and skeletal muscle mitochondria in obese subjects with reduced GH secretion in more detail. Fifteen abdominally obese men with reduced GH secretion were treated for 12 weeks with recombinant human GH. Subjects underwent (31)P-magnetic resonance spectroscopy to assess phosphocreatine (PCr) recovery as an in vivo measure of skeletal muscle mitochondrial function and percutaneous muscle biopsies to assess mRNA expression of IGF-1 and mitochondrial-related genes at baseline and 12 weeks. At baseline, skeletal muscle IGF-1 mRNA expression was significantly associated with PCr recovery (r = 0.79; P = .01) and nuclear respiratory factor-1 (r = 0.87; P = .001), mitochondrial transcription factor A (r = 0.86; P = .001), peroxisome proliferator-activated receptor (PPAR)γ (r = 0.72; P = .02), and PPARα (r = 0.75; P = .01) mRNA expression, and trended to an association with PPARγ coactivator 1-α (r = 0.59; P = .07) mRNA expression. However, serum IGF-1 concentration was not associated with PCr recovery or any mitochondrial gene expression (all P > .10). Administration of recombinant human GH increased both serum IGF-1 (change, 218 ± 29 μg/L; P < .0001) and IGF-1 mRNA in muscle (fold change, 2.1 ± 0.3; P = .002). Increases in serum IGF-1 were associated with improvements in total body fat (r = -0.53; P = .04), trunk fat (r = -0.55; P = .03), and lean mass (r = 0.58; P = .02), but not with PCr recovery (P > .10). Conversely, increase in muscle IGF-1 mRNA was associated with improvements in PCr recovery (r = 0.74; P = .02), but not with body composition parameters (P > .10). These data demonstrate a novel association of skeletal muscle mitochondria with muscle IGF-1 mRNA expression, but independent of serum IGF-1 concentrations.

Relationship Between Serum IGF-1 and Skeletal Muscle IGF-1 mRNA Expression to Phosphocreatine Recovery After Exercise in Obese Men With Reduced GH

PubMed Central

Hamarneh, Sulaiman R.; Murphy, Caitlin A.; Shih, Cynthia W.; Frontera, Walter; Torriani, Martin; Irazoqui, Javier E.

2015-01-01

Context: GH and IGF-1 are believed to be physiological regulators of skeletal muscle mitochondria. Objective: The objective of this study was to examine the relationship between GH/IGF-1 and skeletal muscle mitochondria in obese subjects with reduced GH secretion in more detail. Design: Fifteen abdominally obese men with reduced GH secretion were treated for 12 weeks with recombinant human GH. Subjects underwent 31P-magnetic resonance spectroscopy to assess phosphocreatine (PCr) recovery as an in vivo measure of skeletal muscle mitochondrial function and percutaneous muscle biopsies to assess mRNA expression of IGF-1 and mitochondrial-related genes at baseline and 12 weeks. Results: At baseline, skeletal muscle IGF-1 mRNA expression was significantly associated with PCr recovery (r = 0.79; P = .01) and nuclear respiratory factor-1 (r = 0.87; P = .001), mitochondrial transcription factor A (r = 0.86; P = .001), peroxisome proliferator-activated receptor (PPAR)γ (r = 0.72; P = .02), and PPARα (r = 0.75; P = .01) mRNA expression, and trended to an association with PPARγ coactivator 1-α (r = 0.59; P = .07) mRNA expression. However, serum IGF-1 concentration was not associated with PCr recovery or any mitochondrial gene expression (all P > .10). Administration of recombinant human GH increased both serum IGF-1 (change, 218 ± 29 μg/L; P < .0001) and IGF-1 mRNA in muscle (fold change, 2.1 ± 0.3; P = .002). Increases in serum IGF-1 were associated with improvements in total body fat (r = −0.53; P = .04), trunk fat (r = −0.55; P = .03), and lean mass (r = 0.58; P = .02), but not with PCr recovery (P > .10). Conversely, increase in muscle IGF-1 mRNA was associated with improvements in PCr recovery (r = 0.74; P = .02), but not with body composition parameters (P > .10). Conclusion: These data demonstrate a novel association of skeletal muscle mitochondria with muscle IGF-1 mRNA expression, but independent of serum IGF-1 concentrations. PMID:25375982

Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

NASA Technical Reports Server (NTRS)

Fitzgerald, J.; Hughes-Fulford, M.

1996-01-01

Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.; Gilbertson, V.

1999-01-01

The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

A snoRNA modulates mRNA 3′ end processing and regulates the expression of a subset of mRNAs

PubMed Central

Huang, Chunliu; Shi, Junjie; Guo, Yibin; Huang, Weijun; Huang, Shanshan; Ming, Siqi; Wu, Xingui; Zhang, Rui; Ding, Junjun; Zhao, Wei; Jia, Jie; Huang, Xi; Xiang, Andy Peng

2017-01-01

Abstract mRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing. PMID:28911119

The presence of HBV mRNA in the fertilized in vitro embryo of HBV patients confirms vertical transmission of HBV via the ovum.

PubMed

Ye, F; Jin, Y; Kong, Y; Shi, J Z; Qiu, H T; Zhang, X; Zhang, S L; Lin, S M

2013-05-01

This study aimed to confirm that vertical transmission of hepatitis B virus (HBV) can occur via the infected ovum. Specimens studied were obtained from discarded test-tube embryos from mothers with chronic HBV infection who had received in vitro fertilization treatment. Single-cell reverse transcriptase-polymerase chain reaction was used to detect HBV mRNA in the embryos. HBV mRNA was detected in the cleavage embryos of patients with chronic HBV infection, with a detection rate of 13.2% (5/38). The level of serum HBV DNA was not related to the HBV mRNA positivity rates in embryos. In this study, HBV mRNA was detected in test-tube embryos from HBV-infected mothers who had received in vitro fertilization treatment. This confirms the theory of vertical transmission of HBV via the ovum, thereby providing an important theoretical basis for further study on the mechanism of HBV vertical transmission, influencing factors and blocking measures.

Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

PubMed

Rubenstein, R C; Lyons, B M

2001-07-01

Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovich, B.; Barrieux, A.; Dillmann, W.H.

1987-05-01

Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4more » wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.« less

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Changes in beta-actin mRNA expression in remodeling canine myocardium.

PubMed

Carlyle, W C; Toher, C A; Vandervelde, J R; McDonald, K M; Homans, D C; Cohn, J N

1996-01-01

Beta-actin, a cytoskeletal protein important in the maintenance of cytoarchitecture, has long been thought to be expressed constitutively in myocardial tissue. As such, beta-actin mRNA has been used as a control gene in a wide range of experiments. However, we have uncovered consistent changes in beta-actin mRNA expression in canine myocardium remodeling as a result of insult to the left ventricle. The experimental canine models used were either DC shock damage to the left ventricle or volume overload resulting from severe mitral regurgitation. The remodeling process in both canine models is characterized by an increase in left ventricular mass. PCR amplification using primers designed to selectively amplify the 3' end and a portion of the 3' untranslated region of beta-actin mRNA resulted in the generation of a 297 base pair product predominant only in normal canine myocardium and a 472 base pair product that became increasingly prominent from 1 to 30 days after DC shock damage to the left ventricle and from 10 to 90 days after creation of mitral regurgitation. Northern analysis showed a three-fold increase in beta-actin mRNA after either DC shock or creation of mitral regurgitation. Western analysis revealed an early increase in beta-actin protein followed by an apparent decrease to below baseline levels. These observations suggest that changes in beta-actin mRNA expression accompany the structural alterations that occur in response to myocardial damage. Whether or not the changes in beta-actin mRNA expression play a role in mediating these structural alterations remains to be determined.

Characteristics and Applications of the ToxRefDB In Vivo Datasets from Chronic, Reproductive and Developmental Assays

EPA Science Inventory

ToxRefDB was developed to store data from in vivo animal toxicity studies. The initial focus was populating ToxRefDB with pesticide registration toxicity data that has been historically stored as hard-copy and scanned documents by the Office of Pesticide Programs. A significant p...

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

PubMed

Li, Ping; Noegel, Angelika A

2015-11-16

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Changes in the mRNA expression of structural proteins, hormone synthesis and secretion from bovine placentome sections after DDT and DDE treatment.

PubMed

Wojciechowska, A; Mlynarczuk, J; Kotwica, J

2017-01-15

Disorders in the barrier function and secretory activity of the placenta can be caused by xenobiotics (XB) present in the environment and their accumulation in tissues of living organisms. Thus, the aim of this study was to investigate the effect of 1,1,1-trichloro-2,2,-bis-4-chlorophenyl-ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis-4-chlorophenyl-ethene (DDE) (for 24 or 48h) at doses of 1, 10 or 100ng/ml on the function of cow placentome sections in the second trimester of pregnancy. DDT and DDE affected neither (P>0.05) the viability nor hypoxia inducible factor 1 (HIF1α) mRNA expression of the sections. XB decreased (P<0.05) connexin (Cx) 26, 32, 43 and placenta-specific 1 (PLAC-1) mRNA expression but did not affect (P>0.05) keratin 8 (KRT8) mRNA expression. DDT and DDE also reduced (P<0.05) prostaglandin F2α (PGF2α) synthase (PGFS) mRNA expression, while DDT increased (P<0.05) prostaglandin E2 (PGE2) synthase (PGES) mRNA expression. Neither cyclooxygenase 2 (COX-2) mRNA expression nor PGF2α and PGE2 secretion were affected. Both DDT and DDE increased (P<0.05) neurophysin I/oxytocin (NP1/OT) mRNA expression and oxytocin (OT), oestradiol (E2) and progesterone (P4) secretion while DDT stimulated only 3β-hydroxysteroid dehydrogenase (3βHSD) and cholesterol side-chain cleavage enzyme (CYP11A1) mRNA expression (P<0.05). In summary, DDT and DDE impaired the barrier function and secretory activity of the placenta. Thus, these compounds can disrupt trophoblast invasion, myometrium contractility and gas/nutrient exchange throughout pregnancy in cows. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The induced RNA-binding protein, HuR, targets 3'-UTR region of IL-6 mRNA and enhances its stabilization in periodontitis.

PubMed

Ouhara, K; Munenaga, S; Kajiya, M; Takeda, K; Matsuda, S; Sato, Y; Hamamoto, Y; Iwata, T; Yamasaki, S; Akutagawa, K; Mizuno, N; Fujita, T; Sugiyama, E; Kurihara, H

2018-06-01

RNA-binding proteins (RBPs) regulate mRNA stability by binding to the 3'-untranslated region (UTR) region of mRNA. Human antigen-R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)-6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL-6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in-vitro and in-vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)-derived from Pg (PgLPS) and tumour necrosis factor (TNF)-α in OBA-9, an immortalized human gingival epithelial cell. The luciferase activity of 3'-UTR of IL-6 mRNA was increased by TNF-α, Pg and PgLPS in OBA-9. Luciferase activity was also increased in HuR-over-expressing OBA-9 following a bacterial stimulation. Down-regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL-6. In contrast, the over-expression of HuR increased IL-6 mRNA expression and production in OBA-9. The HuR inhibitor, quercetin, suppressed Pg-induced HuR mRNA expression and IL-6 production in OBA-9. An oral inoculation with quercetin also inhibited bone resorption in ligature-induced periodontitis model mice as a result of down-regulation of IL-6. These results show that HuR modulates inflammatory responses by regulating IL-6. © 2018 British Society for Immunology.

A General Framework for Interrogation of mRNA Stability Programs Identifies RNA-Binding Proteins that Govern Cancer Transcriptomes.

PubMed

Perron, Gabrielle; Jandaghi, Pouria; Solanki, Shraddha; Safisamghabadi, Maryam; Storoz, Cristina; Karimzadeh, Mehran; Papadakis, Andreas I; Arseneault, Madeleine; Scelo, Ghislaine; Banks, Rosamonde E; Tost, Jorg; Lathrop, Mark; Tanguay, Simon; Brazma, Alvis; Huang, Sidong; Brimo, Fadi; Najafabadi, Hamed S; Riazalhosseini, Yasser

2018-05-08

Widespread remodeling of the transcriptome is a signature of cancer; however, little is known about the post-transcriptional regulatory factors, including RNA-binding proteins (RBPs) that regulate mRNA stability, and the extent to which RBPs contribute to cancer-associated pathways. Here, by modeling the global change in gene expression based on the effect of sequence-specific RBPs on mRNA stability, we show that RBP-mediated stability programs are recurrently deregulated in cancerous tissues. Particularly, we uncovered several RBPs that contribute to the abnormal transcriptome of renal cell carcinoma (RCC), including PCBP2, ESRP2, and MBNL2. Modulation of these proteins in cancer cell lines alters the expression of pathways that are central to the disease and highlights RBPs as driving master regulators of RCC transcriptome. This study presents a framework for the screening of RBP activities based on computational modeling of mRNA stability programs in cancer and highlights the role of post-transcriptional gene dysregulation in RCC. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Experimental Regional Ensemble Forecast System (ExREF): Its Use in NWS Forecast Operations and Preliminary Verification

NASA Technical Reports Server (NTRS)

Reynolds, David; Rasch, William; Kozlowski, Daniel; Burks, Jason; Zavodsky, Bradley; Bernardet, Ligia; Jankov, Isidora; Albers, Steve

2014-01-01

The Experimental Regional Ensemble Forecast (ExREF) system is a tool for the development and testing of new Numerical Weather Prediction (NWP) methodologies. ExREF is run in near-realtime by the Global Systems Division (GSD) of the NOAA Earth System Research Laboratory (ESRL) and its products are made available through a website, an ftp site, and via the Unidata Local Data Manager (LDM). The ExREF domain covers most of North America and has 9-km horizontal grid spacing. The ensemble has eight members, all employing WRF-ARW. The ensemble uses a variety of initial conditions from LAPS and the Global Forecasting System (GFS) and multiple boundary conditions from the GFS ensemble. Additionally, a diversity of physical parameterizations is used to increase ensemble spread and to account for the uncertainty in forecasting extreme precipitation events. ExREF has been a component of the Hydrometeorology Testbed (HMT) NWP suite in the 2012-2013 and 2013-2014 winters. A smaller domain covering just the West Coast was created to minimize band-width consumption for the NWS. This smaller domain has and is being distributed to the National Weather Service (NWS) Weather Forecast Office and California Nevada River Forecast Center in Sacramento, California, where it is ingested into the Advanced Weather Interactive Processing System (AWIPS I and II) to provide guidance on the forecasting of extreme precipitation events. This paper will review the cooperative effort employed by NOAA ESRL, NASA SPoRT (Short-term Prediction Research and Transition Center), and the NWS to facilitate the ingest and display of ExREF data utilizing the AWIPS I and II D2D and GFE (Graphical Software Editor) software. Within GFE is a very useful verification software package called BoiVer that allows the NWS to utilize the River Forecast Center's 4 km gridded QPE to compare with all operational NWP models 6-hr QPF along with the ExREF mean 6-hr QPF so the forecasters can build confidence in the use of the

Factors of transforming growth factor beta signalling are co-regulated in human hepatocellular carcinoma.

PubMed

Longerich, Thomas; Breuhahn, Kai; Odenthal, Margarete; Petmecky, Katharina; Schirmacher, Peter

2004-12-01

Transforming growth factor beta (TGFbeta) is a central mitoinhibitory factor for epithelial cells, and alterations of TGFbeta signalling have been demonstrated in many different human cancers. We have analysed human hepatocellular carcinomas (HCCs) for potential pro-tumourigenic alterations in regard to expression of Smad4 and mutations and expression changes of the pro-oncogenic transcriptional co-repressors Ski and SnoN, as well as mRNA levels of matrix metalloproteinase-2 (MMP2), which is transcriptionally regulated by TGFbeta. Smad4 mRNA was detected in all HCCs; while, using immunohistology, loss of Smad4 expression was found in 10% of HCCs. Neither mutations in the transformation-relevant sequences nor significant pro-tumourigenic expression changes of the Ski and SnoN genes were detected. In HCC cell lines, expression of both genes was regulated, potentially involving phosphorylation. Ski showed a distinct nuclear speckled pattern, indicating recruitment to active transcription complexes. MMP2 mRNA levels were increased in 19% of HCCs, whereas MMP2 mRNA was not detectable in HCC cell lines, suggesting that MMP2 was derived only from tumour stroma cells. Transcript levels of Smad4, Ski, SnoN and MMP2 correlated well. These data argue against a significant role of Ski and SnoN in human hepatocarcinogenesis and suggest that, in the majority of HCCs, the analysed factors are co-regulated by an upstream mechanism, potentially by TGFbeta itself.

Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

PubMed Central

Pascale, Alessia; Amadio, Marialaura; Scapagnini, Giovanni; Lanni, Cristina; Racchi, Marco; Provenzani, Alessandro; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

2005-01-01

More than 1 in 20 human genes bear in the mRNA 3′ UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCα isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCα abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCα-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role. PMID:16099831

Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma.

PubMed Central

Kitadai, Y.; Ellis, L. M.; Tucker, S. L.; Greene, G. F.; Bucana, C. D.; Cleary, K. R.; Takahashi, Y.; Tahara, E.; Fidler, I. J.

1996-01-01

We examined the expression level of several genes that regulate different steps of metastasis in formalin-fixed, paraffin-embedded archival specimens of primary human colon carcinomas from patients with at least 5 years of follow-up. The expression of epidermal growth factor receptor, basic fibroblast growth factor, type IV collagenase, E-cadherin, and multidrug resistance (mdr-1) was examined by a colorimetric in situ mRNA hybridization technique concentrating on reactivity at the periphery of the neoplasms. The in situ hybridization technique revealed inter- and intratumor heterogeneity for expression of the metastasis-related genes. The expression of basic fibroblast growth factor, collagenase type IV, epidermal growth factor receptor, and mdr-1 mRNA was higher in Dukes's stage D than in Dukes' stage B tumors. Among the 22 Dukes' stage B neoplasms, 5 specimens exhibited a high expression level of epidermal growth factor receptor, basic fibroblast growth factor, and collagenase type IV. Clinical outcome data (5-year follow-up) revealed that all 5 patients with Dukes' stage B tumors developed distant metastasis (recurrent disease), whereas the other 17 patients with Dukes' stage B tumors expressing low levels of the metastasis-related genes were disease-free. Multivariate analysis identified high levels of expression of collagenase type IV and low levels of expression of E-cadherin as independent factors significantly associated with metastasis or recurrent disease. More specifically, metastatic or recurrent disease was associated with a high ratio (> 1.35) of expression of collagenase type IV to E-cadherin (specificity of 95%). Collectively, the data show that multiparametric in situ hybridization analysis for several metastasis-related genes may predict the metastatic potential, and hence the clinical outcome, of individual lymph-node-negative human colon cancers. Images Figure 1 Figure 2 PMID:8909244

A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

PubMed

Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R

2014-02-01

Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents

PubMed Central

Nagata, Seigo; Hamasaki, Tomohiro; Uetake, Koichi; Masuda, Hirofumi; Takagaki, Kazuchika; Oka, Natsuhisa; Wada, Takeshi; Ohgi, Tadaaki; Yano, Junichi

2010-01-01

Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2′-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5′-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5′-cap and a 3′-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications. PMID:20660478

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

PubMed Central

Eiring, Anna M.; Harb, Jason G.; Neviani, Paolo; Garton, Christopher; Oaks, Joshua J.; Spizzo, Riccardo; Liu, Shujun; Schwind, Sebastian; Santhanam, Ramasamy; Hickey, Christopher J.; Becker, Heiko; Chandler, Jason C.; Andino, Raul; Cortes, Jorge; Hokland, Peter; Huettner, Claudia S.; Bhatia, Ravi; Roy, Denis C.; Liebhaber, Stephen A.; Caligiuri, Michael A.; Marcucci, Guido; Garzon, Ramiro; Croce, Carlo M.; Calin, George A.; Perrotti, Danilo

2010-01-01

SUMMARY MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA’s seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins. PMID:20211135

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation.

PubMed

Eswari, S; Sai Kumar, G; Sharma, G Taru

2013-05-01

Summary The objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus-oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8-16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

PubMed

Crisan, D; Anstett, M J

1995-07-01

Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

Integrative comparison of mRNA expression patterns in breast cancers from Caucasian and Asian Americans with implications for precision medicine

PubMed Central

Wang, Jianan; He, Max M; Li, Liren; Zhang, Jinfeng

2016-01-01

Asian Americans (AS) have significantly lower incidence and mortality rates of breast cancer (BRCA) than Caucasian Americans (CA). While this racial disparity has been documented the underlying pathogenetic factors explaining it are obscure. We addressed this issue by an integrative genomics approach to compare mRNA expression between AS and CA cases of BRCA. RNA-seq data from the Cancer Genome Atlas showed that mRNA expression revealed significant differences at gene and pathway levels. Increased susceptibility and severity in CA patients were likely the result of synergistic environmental and genetic risk factors, with arachidonic acid metabolism and PPAR signaling pathways implicated in linking environmental and genetic factors. An analysis that also added eQTL data from the Genotype-Tissue Expression Project and single nucleotide polymorphism (SNP) data from the 1000 Genomes Project identified several SNPs associated with differentially expressed genes. Overall, the associations we identified may enable a more focused study of genotypic differences that may help explain the disparity in BRCA incidence and mortality rates in CA and AS populations and inform precision medicine. PMID:28069798

Exaptive origins of regulated mRNA decay in eukaryotes

PubMed Central

Hamid, Fursham M.

2016-01-01

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.

PubMed

Pillai, Ramesh S; Artus, Caroline G; Filipowicz, Witold

2004-10-01

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA. Copyright 2004 RNA Society

Arc mRNA induction in striatal efferent neurons associated with response learning.

PubMed

Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

2007-07-01

The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3

PubMed Central

Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal GP; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

2015-01-01

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. PMID:26637431

Increased expression of cyclin B1 mRNA coincides with diminished G{sub 2}-phase arrest in irradiated HeLa cells treated with staurosporine or caffeine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernhard, E.J.; Maity, A.; McKenna, W.G.

1994-12-01

The irradiation of cells results in delayed progression through the G{sub 2} phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G{sub 2}-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G{sub 2}-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2ng/ml) staurosporine, which releases the radiation-induced G{sub 2}-phase arrest. In HeLamore » cells, the G{sub 2}-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G{sub 2}-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G{sub 2}-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury. 33 refs., 6 figs.« less

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

PubMed

Lee, Hyung Chul; Jung, Seung Hee; Hwang, Hyun Jung; Kang, Donghee; De, Supriyo; Dudekula, Dawood B; Martindale, Jennifer L; Park, Byungkyu; Park, Seung Kuk; Lee, Eun Kyung; Lee, Jeong-Hwa; Jeong, Sunjoo; Han, Kyungsook; Park, Heon Joo; Ko, Young-Gyu; Gorospe, Myriam; Lee, Jae-Seon

2017-06-20

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effects of added fermentable carbohydrates in the diet on intestinal proinflammatory cytokine-specific mRNA content in weaning piglets.

PubMed

Pié, S; Awati, A; Vida, S; Falluel, I; Williams, B A; Oswald, I P

2007-03-01

There is increasing evidence showing that dietary supplementation with prebiotics can be effective in the treatment of intestinal inflammation. Because weaning time is characterized by rapid intestinal inflammation, this study investigated the effect of a diet supplemented with a combination of 4 fermentable carbohydrates (lactulose, inulin, sugarbeet pulp, and wheat starch) on the mRNA content of proinflammatory cytokines in newly weaned piglets. Cytokines (IL-1beta, IL-6, IL-8, IL-12p40, IL-18, and tumor necrosis factor-alpha) were analyzed using a semiquantitative reverse-transcription PCR technique on d 1, 4, and 10 in the ileum and colon of piglets fed either a test diet (CHO) or a control diet. In addition to the diet, the effect of enforced fasting on cytokine mRNA content was also evaluated. No effect of fasting was observed on the pro-inflammatory cytokine mRNA content. Our results showed that the CHO diet induced an up-regulation of IL-6 mRNA content in the colon of piglets 4 d postweaning. This up-regulation was specific for the animals fed the CHO diet and was not observed in animals fed the control diet. An increase in IL-1beta mRNA content was also observed on d 4 postweaning in all of the piglets. Correlations between proinflammatory cytokines and the end-products of fermentation indicated that the regulation of cytokines may be linked with some of the fermentation end-products such as branched-chain fatty acids, which are in turn end-products of protein fermentation.

The role of mammalian Staufen on mRNA traffic: a view from its nucleocytoplasmic shuttling function.

PubMed

Miki, Takashi; Takano, Keizo; Yoneda, Yoshihiro

2005-01-01

The localization of mRNA in neuronal dendrites plays a role in both locally and temporally regulated protein synthesis, which is required for certain forms of synaptic plasticity. RNA granules constitute a dendritic mRNA transport machinery in neurons, which move along microtubules. RNA granules contain densely packed clusters of ribosomes, but lack some factors that are required for translation, suggesting that they are translationally incompetent. Recently some of the components of RNA granules have been identified, and their functions are in the process of being examined, in attempts to better understand the properties of RNA granules. Mammalian Staufen, a double-stranded RNA binding protein, is a component of RNA granules. Staufen is localized in the somatodendritic domain of neurons, and plays an important role in dendritic mRNA targeting. Recently, one of the mammalian homologs of Staufen, Staufen2 (Stau2), was shown to shuttle between the nucleus and the cytoplasm. This finding suggests the possibility that Stau2 binds RNA in the nucleus and that this ribonucleoprotein particle is transported from the nucleus to RNA granules in the cytoplasm. A closer study of this process might provide a clue to the mechanism by which RNA granules are formed.

Selective decline of Nogo mRNA in the aging brain.

PubMed

Trifunovski, Alexandra; Josephson, Anna; Bickford, Paula C; Olson, Lars; Brené, Stefan

2006-06-26

The Nogo system has recently been implicated not only in regeneration but also in modulating plasticity. One reason for declining memory functions in aging may be altered plasticity in the aged hippocampus and cortex cerebri. Therefore, we have examined the levels of mRNA encoding Nogo, OMgp and MAG, as well as the receptor components NgR, Lingo-1 and Troy in cortex and hippocampus of young (4 months), middle aged (16 months) and old (24 months) Fisher 344 rats. No significant changes of receptor components or the ligands OMgp or MAG were observed. Nogo mRNA, however, was significantly decreased in hippocampal subregions of aged animals. The specific decrease of Nogo mRNA levels in hippocampus and possibly cortex cerebri may relate to age-dependent decline of brain plasticity.

RNA-Binding Proteins Revisited - The Emerging Arabidopsis mRNA Interactome.

PubMed

Köster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-06-01

RNA-protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture - where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Paraoxonase-2 and paraoxonase-3: comparison of mRNA expressions in the placentae of unexplained intrauterine growth restricted and noncomplicated pregnancies.

PubMed

Dikbas, Levent; Yapca, Omer Erkan; Dikbas, Neslihan; Gundogdu, Cemal

2017-05-01

Recent evidence suggests that oxidative stress is involved in the pathophysiology of many human diseases. It has been demonstrated that oxidative stress is associated with intrauterine growth restriction (IUGR), and the depletion of placental antioxidant systems has been suggested as a key factor in this disease. Our aims were to explore the possible role of antioxidant paraoxonase-2 (PON2) and paraoxonase-3 (PON3) in the pathophysiology of unexplained IUGR. We have studied the expression of mRNA for PON2, PON3 in placental tissues by using RT-qPCR. Two groups, consisting of normal (n = 18) and unexplained IUGR pregnancies (n = 20) were compared. Our results demonstrated that there were no significant differences in the mRNA expressions of PON2, PON3 between the two groups (p = 0.28, p = 0.90, respectively). PON2 and PON3 were down-regulated in IUGR. Antenatal steroid therapy had no effect on the expression mRNA in placentae of unexplained IUGR pregnancies compared to non-treated group. These results suggest that PON2, PON3 mRNA levels were not changed significantly in placentae of IUGR when compared to normal pregnant women.

Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride.

PubMed

Tian, Yuhu; Huo, Meijun; Li, Guangsheng; Li, Yanyan; Wang, Jundong

2016-10-01

F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

The expression of Apoc3 mRNA is regulated by HNF4α and COUP-TFII, but not acute retinoid treatments, in primary rat hepatocytes and hepatoma cells.

PubMed

Howell, Meredith; Li, Rui; Zhang, Rui; Li, Yang; Chen, Wei; Chen, Guoxun

2014-02-01

Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoic acid (RA) has been attributed to the induction of apolipoprotein C-III expression. To understand the role of retinoids in the development of hyperlipidemia in ZF rats, the expression levels of several selected RA-responsive genes in the liver and isolated hepatocytes from Zucker lean (ZL) and ZF rats were compared using real-time PCR. The Rarb and Srebp-1c mRNA levels are higher in the liver and isolated hepatocytes from ZF than ZL rats. The Apoc3 mRNA level is only higher in the isolated hepatocytes from ZF than ZL rats. To determine whether dynamic RA production acutely regulates Apoc3 expression, its mRNA levels in response to retinoid treatments or adenovirus-mediated overexpression of hepatocyte nuclear factor 4 alpha (HNF4α) and chicken ovalbumin upstream-transcription factor II (COUP-TFII) were analyzed. Retinoid treatments for 2-6 h did not induce the expression of Apoc3 mRNA. The overexpression of HNF4α or COUP-TFII induced or inhibited Apoc3 expression, respectively. We conclude that short-term retinoid treatments could not induce Apoc3 mRNA expression, which is regulated by HNF4α and COUP-TFII in hepatocytes.

Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen.

PubMed

Werme, M; Thorén, P; Olson, L; Brené, S

2000-08-01

Physical activities such as long-distance running can be habit forming and associated with a sense of well-being to a degree that justifies comparison with drug-induced addictive behaviours. To understand molecular similarities and dissimilarities controlling these behaviours in humans we compared the effects of running in running wheels to the effects of chronic cocaine or morphine administration on mRNA levels in brain reward pathways in the inbred Fischer and Lewis rat strains. These strains are both inbred from the Sprague-Dawley strain; Lewis rats display a higher preference towards addictive drugs and running than do Fischer rats. After chronic cocaine or running a similar increase of dynorphin mRNA in medial caudate putamen was found in the Lewis rat, suggesting common neuronal adaptations in this brain region to both cocaine and running. Fischer and Lewis rats both responded to cocaine with increased dynorphin mRNA levels in medial caudate putamen. However, only Lewis rats increased dynorphin mRNA after running, possibly reflecting the much higher degree of running by the Lewis strain as compared to the Fischer strain. Moreover, the running-induced upregulation of dynorphin mRNA was blocked by the opioid receptor antagonist naloxone. We suggest that running increases dynorphin mRNA by a mechanism that involves endogenous opioids. The voluntary wheel-running model in rats might be used to study natural reward and compulsive behaviours and possibly also to screen candidate drugs for treatment of compulsive disorders.

Characterization of a major late herpes simplex virus type 1 mRNA.

PubMed

Costa, R H; Devi, B G; Anderson, K P; Gaylord, B H; Wagner, E K

1981-05-01

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

PubMed Central

Dul, Ed; Mahoney, Michael E.; Wulff, Daniel L.

1987-01-01

Starting with the λ pRE- strain λctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, λdya2 ctr1 cy3008 and λ dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations of cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'.—The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII- mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A → G mutation four bases before the initial AUG codon, and cII3059 , a GUU → GAU (Val2 → Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested. However neither the ctr1 mutation nor the dya2 mutation has much effect on translation efficiency in an otherwise cII+ background, indicating that other factors must limit the rate of translation of cII mRNA under these conditions. PMID:2953647

Development, food intake, and ethinylestradiol influence hepatic triglyceride lipase and LDL-receptor mRNA levels in rats.

PubMed

Staels, B; Jansen, H; van Tol, A; Stahnke, G; Will, H; Verhoeven, G; Auwerx, J

1990-07-01

The influence of development and ethinylestradiol on low density lipoprotein (LDL)-receptor mRNA and hepatic triglyceride lipase (HTGL) activity and mRNA levels was studied in rat liver and intestine. Intestinal LDL-receptor mRNA levels are maximal in the perinatal period, whereas liver LDL-receptor and HTGL mRNA levels are highest after weaning in adult life. All mRNA levels reach a maximum between day 15 and 20 when rats still consume a lipid-rich diet, and increase twofold during weaning. Liver and intestinal LDL-receptor mRNA levels are not influenced by ovariectomy, but increase after ethinylestradiol treatment. Liver LDL-receptor mRNA shows a dose-dependent increase after ethinylestradiol and a sevenfold rise in liver LDL-receptor mRNA is attained with a dose of 2000 micrograms/day. Intestinal LDL-receptor mRNA increases slightly more than twofold after ethinylestradiol and this increase is not dose-dependent. Changes in LDL-receptor mRNA are independent of changes in food intake induced by ethinylestradiol treatment, since they are still observed after pair-feeding. The ethinylestradiol-induced increases in LDL-receptor mRNA levels are reflected by decreased serum apoB levels. HTGL mRNA levels increase after ovariectomy and show a dose-dependent decrease after ethinylestradiol. Pair-feeding abolishes the increase seen after ovariectomy, while the estrogen-mediated decrease is attenuated. These alterations in HTGL mRNA are reflected by similar changes in liver HTGL activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

PubMed

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

Distribution of cholecystokinin mRNA and peptides in the human brain.

PubMed

Lindefors, N; Brené, S; Kopp, J; Lindén, A; Brodin, E; Sedvall, G; Persson, H

1991-01-01

Expression of preprocholecystokinin mRNA was studied in regions of post mortem human brain using RNA blot analysis (Northern blot) and in situ hybridization. Northern blot analysis using a cDNA probe showed high levels of an approximately 0.8 kb preprocholecystokinin mRNA in all regions of neocortex examined. Lower levels of preprocholecystokinin mRNA were detected in amygdaloid body and thalamus. In situ hybridization analysis using the same cDNA probe revealed numerous weakly labelled neurons in different areas of human neocortex and less numerous neurons in hippocampus and amygdaloid body. High-performance liquid-chromatography and gel-chromatography combined with radioimmunoassay of cholecystokinin-like immunoreactivity from human cerebral cortex and caudate nucleus revealed two major forms, one coeluting with sulphated cholecystokinin-8 and the other coeluting with sulphated cholecystokinin-58. Two minor components coeluting with cholecystokinin-4 and cholecystokinin-5 were also detected. The finding of cholecystokinin-like immunoreactivity corresponding to cholecystokinin-8 and cholecystokinin-58 in caudate nucleus where no preprocholecystokinin mRNA was found, indicates the presence of these peptides in afferent nerve terminals.

Quantitative imaging of single mRNA splice variants in living cells

NASA Astrophysics Data System (ADS)

Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

2014-06-01

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

ToxRefDB 2.0: Improvements in Capturing Qualitative and Quantitative Data from in vivo Toxicity Studies (SOT)

EPA Science Inventory

The Toxicity Reference Database (ToxRefDB) is a publicly accessible resource that contains 40+ years of in vivo dose-response toxicological studies. ToxRefDB provides curated in vivo toxicity data for systematic evaluation of a continuously expanding catalog of chemicals, and co...

Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving.

PubMed

Theberge, Florence R M; Pickens, Charles L; Goldart, Evan; Fanous, Sanya; Hope, Bruce T; Liu, Qing-Rong; Shaham, Yavin

2012-12-01

Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial prefrontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. We trained rats to self-administer heroin or saline for 9-10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone's (0-1.0 mg/kg) effect on extinction responding after 1 and 15 days. Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving.

Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving

PubMed Central

Theberge, Florence R. M.; Pickens, Charles L.; Goldart, Evan; Fanous, Sanya; Hope, Bruce T.; Liu, Qing-Rong

2013-01-01

Rationale and objectives Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial pre-frontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. Methods We trained rats to self-administer heroin or saline for 9–10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone’s (0–1.0 mg/kg) effect on extinction responding after 1 and 15 days. Results Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Conclusions Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving. PMID:22790874

Cykotine mRNA expression in mouse retina after laser injury by reverse transcriptase-polymerase chain reaction (RT-PCR)

NASA Astrophysics Data System (ADS)

Schuschereba, Steven T.; Bowman, Phillip D.; Ujimore, Veronica; Hoxie, Stephen W.; Pizarro, Jose M.; Cross, Michael E.; Lund, David J.

1996-04-01

The purpose of this study was to identify cytokines produced by the retina after laser injury. With the aid of a scanning laser ophthalmoscope (SLO), right eyes of mice received lesions from a continuous wave argon laser. Left eyes served as unirradiated controls. At 2, 4, 6, 12, 24, and 48 hr after laser irradiation groups of 3 mice were euthanized and retinas fixed for histology or isolated for RNA. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) and subjected to polymerase chain reaction for the following cytokines: tumor necrosis factor-(alpha) (TNF-(alpha) ), interleukin-1(alpha) /(Beta) (IL- 1(alpha) /(Beta) ), interleukin-6 (IL-6), transforming growth factor-(Beta) 1 (TGF- (Beta) 1), macrophage colony stimulating factor (M-CSF), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Histologically, lesions were confined to the photoreceptors, retinal pigment epithelium, and choroid. In laser-injured retinas, mRNA levels were elevated for IL-1(alpha) , TGF-(Beta) 1, iNOS, and G3PDH, but not TNF-(alpha) , IL-1(Beta) , or IL-6. It appears that the retina, in response to laser injury, upregulates a select number of cytokines in a time-course dependent fashion.

Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

PubMed Central

Suzuki, Masataka G.; Ito, Haruka; Aoki, Fugaku

2014-01-01

Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity. PMID:24758924

Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis latex.

PubMed

Wadeesirisak, Kanthida; Castano, Sabine; Berthelot, Karine; Vaysse, Laurent; Bonfils, Frédéric; Peruch, Frédéric; Rattanaporn, Kittipong; Liengprayoon, Siriluck; Lecomte, Sophie; Bottier, Céline

2017-02-01

Rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. The repartition of both proteins on the small or large rubber particles seems to differ, but their role in the irreversible coagulation of the rubber particle is still unknown. In this study we highlighted the different modes of interactions of both recombinant proteins with different classes of lipids extracted from Hevea brasiliensis latex, and defined as phospholipids (PL), glycolipids (GL) and neutral lipids (NL). We combined two biophysical methods, polarization modulated-infrared reflection adsorption spectroscopy (PM-IRRAS) and ellipsometry to elucidate their interactions with monolayers of each class of lipids. REF1 and SRPP1 interactions with native lipids are clearly different; SRPP1 interacts mostly in surface with PL, GL or NL, without modification of its structure. In contrast REF1 inserts deeply in the lipid monolayers with all lipid classes. With NL, REF1 is even able to switch from α-helice conformation to β-sheet structure, as in its aggregated form (amyloid form). Interaction between REF1 and NL may therefore have a specific role in the irreversible coagulation of rubber particles. Copyright © 2016 Elsevier B.V. All rights reserved.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

PubMed Central

Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-01-01

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

PubMed

Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

2013-01-01

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (P<0.01). To our knowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3.

PubMed

Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal G P; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

2015-12-04

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. Copyright © 2016 Roux et al.

Food restriction in young Japanese quails: effects on growth, metabolism, plasma thyroid hormones and mRNA species in the thyroid hormone signalling pathway.

PubMed

Rønning, Bernt; Mortensen, Anne S; Moe, Børge; Chastel, Olivier; Arukwe, Augustine; Bech, Claus

2009-10-01

Young birds, in their post-natal growth period, may reduce their growth and metabolism when facing a food shortage. To examine how such responses can be mediated by endocrine-related factors, we exposed Japanese quail chicks to food restriction for either 2 days (age 6-8 days) or 5 days (age 6-11 days). We then measured growth and resting metabolic rate (RMR), and circulating 3,3',5-triiodo-l-thyronine (T3) and 3,5,3',5'-tetraiodothyronine (T4) levels as well as expression patterns of genes involved in growth (insulin-like growth factor-I: IGF-I) and thyroid hormone signalling (thyroid-stimulating hormone-beta: TSHbeta, type II iodothyronine deiodinase: D2, thyroid hormone receptors isoforms: TRalpha and TRbeta). The food-restricted chicks receiving a weight-maintenance diet showed reductions in structural growth and RMR. Plasma levels of both T3 and T4 were reduced in the food-restricted birds, and within the 5 days food-restricted group there was a positive correlation between RMR and T3. IGF-I mRNA showed significantly higher abundance in the liver of ad libitum fed birds at day 8 compared with food-restricted birds. In the brain, TSHbeta mRNA level tended to be lower in food-restricted quails on day 8 compared with controls. Furthermore, TRalpha expression was lower in the brain of food-restricted birds at day 8 compared with birds fed ad libitum. Interestingly, brain D2 mRNA was negatively correlated with plasma T3 levels, tending to increase with the length of food restriction. Overall, our results show that food restriction produced significant effects on circulating thyroid hormones and differentially affected mRNA species in the thyroid hormone signalling pathway. Thus, we conclude that the effects of food restriction observed on growth and metabolism were partly mediated by changes in the endocrine-related factors investigated.

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish Tie1 mRNA by Long Noncoding RNA During Brain Vascular Assembly.

PubMed

Chowdhury, Tamjid A; Koceja, Chris; Eisa-Beygi, Shahram; Kleinstiver, Benjamin P; Kumar, Suresh N; Lin, Chien-Wei; Li, Keguo; Prabhudesai, Shubhangi; Joung, J Keith; Ramchandran, Ramani

2018-05-03

Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus- tie1 antisense ( tie1AS ), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS . Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS ). © 2018 American Heart Association, Inc.

Detection of circulating Bmi-1 mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancer.

PubMed

Zhang, Xin; Wang, Chuanxin; Wang, Lili; Du, Lutao; Wang, Shun; Zheng, Guixi; Li, Wei; Zhuang, Xuewei; Zhang, Xuhua; Dong, Zhaogang

2012-07-01

Bmi-1 is overexpressed in uterine cervical cancer (UCC) and is found to be associated with adverse clinical characteristics and poor prognosis. However, little information is available on the status of circulating Bmi-1 mRNA in UCC. Because circulating cell-free nucleic acids have emerged as a novel class of markers for cancer detection, our research aims to address this question by detecting the circulating Bmi-1 mRNA and to assess its diagnostic and prognostic potential in UCC. Reverse transcription quantitative real-time PCR method was established to detect the circulating Bmi-1 mRNA in plasma of 109 patients with UCC, 138 patients with cervical intraepithelial neoplasia (CIN) and 80 healthy volunteers, and found that it was significantly increased in UCC compared with CINs and healthy controls (all at p < 0.001). Moreover, its high level was significantly correlated with advanced clinical stage (p < 0.001) and positive lymph nodes metastasis (p = 0.002). The area under the receiver operating characteristic curve (AUC) was 0.881, and the optimal cut-off value was 0.057, providing a sensitivity of 69.7% and a specificity of 95.9%. The AUC for circulating Bmi-1 mRNA showed higher diagnosis capability than that for SCC-Ag (p = 0.035) or CA125 (p < 0.001) currently utilized. Kaplan-Meier analysis demonstrated a correlation between increased circulating Bmi-1 mRNA level and reduced disease-free survival (DFS) (p = 0.001) and overall survival (OS) (p = 0.015). And, Cox analysis indicated that it was an independent prognostic factor for DFS and OS. We conclude that circulating Bmi-1 mRNA may be a potential noninvasive molecular marker for diagnosis and prognosis of UCC. Copyright © 2011 UICC.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

PubMed

Spangler, Jacob B; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

PubMed Central

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

PubMed

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Nonsense-mediated mRNA decay: inter-individual variability and human disease

PubMed Central

Nguyen, Lam Son; Wilkinson, Miles; Gecz, Jozef

2013-01-01

Nonsense-Mediated mRNA Decay (NMD) is a regulatory pathway that functions to degrade transcripts containing premature termination codons (PTCs) and to maintain normal transcriptome homeostasis. Nonsense and frameshift mutations that generate PTCs cause approximately one-third of all known human genetic diseases and thus NMD has a potentially important role in human disease. In genetic disorders in which the affected genes carry PTC-generating mutations, NMD acts as a double-edge sword. While it can benefit the patient by degrading PTC-containing mRNAs that encode detrimental, dominant-negative truncated proteins, it can also make the disease worse when a PTC-containing mRNA is degraded that encodes a mutant but still functional protein. There is evidence that the magnitude of NMD varies between individuals, which, in turn, has been shown to correlate with both clinical presentations and the patients’ responses to drugs that promote read-through of PTCs. In this review, we examine the evidence supporting the existence of inter-individual variability in NMD efficiency and discuss the genetic factors that underlie this variability. We propose that inter-individual variability in NMD efficiency is a common phenomenon in human populations and that an individual’s NMD efficiency should be taken into consideration when testing, developing, and making therapeutic decisions for diseases caused by genes harboring PTCs. PMID:24239855

Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

PubMed

Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

2014-12-16

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

Microinjection and Fluorescence In Situ Hybridization Assay for Studying mRNA Export in Mammalian Cells.

PubMed

Wang, Ke; Shi, Min; Cheng, Hong

2017-01-01

Microinjection and Fluorescence in situ Hybridization (FISH) assay is a useful method for mRNA export studies, which can overcome the problems of traditional transfection in cells. Here, we describe the method of microinjection and FISH assay applied in investigation of mRNA export. By this method we can estimate the mRNA export kinetics, examining mRNA export in cells with low transfection efficiencies, and observing nuclear export of aberrant RNAs.

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

PubMed

Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

2016-06-01

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.