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Sample records for caveolin-1-mediated post-transcriptional regulation

  1. Computational Investigations of Post-Transcriptional Regulation

    DEFF Research Database (Denmark)

    Rasmussen, Simon Horskjær

    and miRNA regulation was studied by cross-linking immunoprecipitation (CLIP) and RBP double knockdown experiments. A comprehensive analysis of 107 CLIP datasets of 49 RBPs demonstrated that RBPs modulate miRNA regulation. Results suggest it is mediated by RBP-binding hotspots that likely...... investigated using high-throughput data. Analysis of IMP RIP-seq, iCLIP and RNA-seq datasets identified transcripts associated with cytoplasmic IMP ribonucleoproteins. Many of these transcripts were functionally involved in actin cytoskeletal remodeling. Further analyses of this data permitted estimation...... of a bipartite motif, composed of an AU-rich and a CA-rich domain. In addition, a regulatory motif discovery method was developed and applied to identify motifs using differential expression data and CLIP-data in the above investigations. This thesis increased the understanding of the role of RBPs in mi...

  2. Mechanisms of post-transcriptional gene regulation in bacterial biofilms

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    Viveka eVadyvaloo

    2014-03-01

    Full Text Available Abstract Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria.

  3. Post-transcriptional regulation of gene expression in Yersinia species

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    Chelsea A Schiano

    2012-11-01

    Full Text Available Proper regulation of gene expression is required by bacterial pathogens to respond to continually changing environmental conditions and the host response during the infectious process. While transcriptional regulation is perhaps the most well understood form of controlling gene expression, recent studies have demonstrated the importance of post-transcriptional mechanisms of gene regulation that allow for more refined management of the bacterial response to host conditions. Yersinia species of bacteria are known to use various forms of post-transcriptional regulation for control of many virulence-associated genes. These include regulation by cis- and trans-acting small non-coding RNAs, RNA-binding proteins, RNases, and thermoswitches. The effects of these and other regulatory mechanisms on Yersinia physiology can be profound and have been shown to influence type III secretion, motility, biofilm formation, host cell invasion, intracellular survival and replication, and more. In this review, we will discuss these and other post-transcriptional mechanisms and their influence on virulence gene regulation, with a particular emphasis on how these processes influence the virulence of Yersinia in the host.

  4. TOR-dependent post-transcriptional regulation of autophagy.

    Science.gov (United States)

    Hu, Guowu; McQuiston, Travis; Bernard, Amélie; Park, Yoon-Dong; Qiu, Jin; Vural, Ali; Zhang, Nannan; Waterman, Scott R; Blewett, Nathan H; Myers, Timothy G; Maraia, Richard J; Kehrl, John H; Uzel, Gulbu; Klionsky, Daniel J; Williamson, Peter R

    2015-01-01

    Regulation of autophagy is required to maintain cellular equilibrium and prevent disease. While extensive study of post-translational mechanisms has yielded important insights into autophagy induction, less is known about post-transcriptional mechanisms that could potentiate homeostatic control. In our study, we showed that the RNA-binding protein, Dhh1 in Saccharomyces cerevisiae and Vad1 in the pathogenic yeast Cryptococcus neoformans is involved in recruitment and degradation of key autophagy mRNAs. In addition, phosphorylation of the decapping protein Dcp2 by the target of rapamycin (TOR), facilitates decapping and degradation of autophagy-related mRNAs, resulting in repression of autophagy under nutrient-replete conditions. The post-transcriptional regulatory process is conserved in both mouse and human cells and plays a role in autophagy-related modulation of the inflammasome product IL1B. These results were then applied to provide mechanistic insight into autoimmunity of a patient with a PIK3CD/p110δ gain-of-function mutation. These results thus identify an important new post-transcriptional mechanism of autophagy regulation that is highly conserved between yeast and mammals.

  5. Role of Sam68 in Post-Transcriptional Gene Regulation

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    Flora Sánchez-Jiménez

    2013-11-01

    Full Text Available The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K homology (KH single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.

  6. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    Science.gov (United States)

    Kula, Anna; Marcello, Alessandro

    2012-01-01

    Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. PMID:24832221

  7. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

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    Alessandro Marcello

    2012-07-01

    Full Text Available Gene expression of the human immunodeficiency virus type 1 (HIV-1 is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE. These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function.

  8. DMPD: Post-transcriptional regulation of proinflammatory proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15075353 Post-transcriptional regulation of proinflammatory proteins. Anderson P, P...hillips K, Stoecklin G, Kedersha N. J Leukoc Biol. 2004 Jul;76(1):42-7. Epub 2004 Apr 1. (.png) (.svg) (.html) (.csml) Show Post...-transcriptional regulation of proinflammatory proteins. PubmedID 15075353 Title Post-tr

  9. Post-transcriptional regulation of the chicken thymidine kinase gene.

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    Groudine, M; Casimir, C

    1984-02-10

    In attempting to understand the molecular basis of the control of chicken thymidine kinase (cTK) gene expression, we have examined the steady state cTK RNA content, and the patterns of DNA methylation, chromatin structure and endogenous nuclear runoff transcription of this gene in dividing and non-dividing cells. Our results reveal that the steady state level of cTK poly A+ RNA is correlated with the divisional activity of normal avian cells and tissues. However, no differences in the pattern of Hpa II site methylation or chromatin structure are found among cells containing high or undetectable levels of steady state cTK RNA. In addition, no differences in cTK transcription as assayed by nuclear runoff experiments are detectable in isolated nuclei derived from dividing or non-dividing cells containing high or low levels of steady state cTK RNA. These results suggest that the principal control of chicken thymidine kinase gene expression is post-transcriptional in nature.

  10. The 5th Symposium on Post-Transcriptional Regulation of Plant Gene Expression (PTRoPGE)

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning; Marie Petrocek; Bonnie Bartel

    2006-06-01

    The 5th Symposium on Post-Transcriptional Regulation of Plant Gene Expression (PTRoPGE) will be held June 8-12, 2005 at the University of Texas at Austin. Exciting new and ongoing discoveries show significant regulation of gene expression occurs after transcription. These post-transcriptional control events in plants range from subtle regulation of transcribed genes and phosphorylation, to the processes of gene regulation through small RNAs. This meeting will focus on the regulatory role of RNA, from transcription, through translation and finally degradation. The cross-disciplinary design of this meeting is necessary to encourage interactions between researchers that have a common interest in post-transcriptional gene expression in plants. By bringing together a diverse group of plant molecular biologist and biochemists at all careers stages from across the world, this meeting will bring about more rapid progress in understanding how plant genomes work and how genes are finely regulated by post-transcriptional processes to ultimately regulate cells.

  11. Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

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    John Martin

    Full Text Available Transforming Growth Factor Beta-1 (TGF-β1 is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-β1 is post-transcriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-β1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular cells in the context of diabetic nephropathy. In this study, we have investigated the role of the TGF-β1 3'Untranslated Region (3'UTR. Two different 3'UTR lengths have been reported for TGF-β1, of 543 and 137 nucleotides. Absolute quantification showed that, while both UTR lengths were detectable in various human cell types and in a broad range of tissues, the short form predominated in the kidney and elsewhere. Expression of both forms was up-regulated following auto-induction by TGF-β1, but the short:long UTR ratio remained constant. Incorporation of the short UTR into a luciferase reporter vector significantly reduced reporter protein synthesis without major effect on RNA amount, suggesting post-transcriptional inhibition. In silico approaches identified multiple binding sites for miR-744 located in the proximal TGF-β1 3'UTR. A screen in RNA from human tissues showed widespread miR-744 expression. miR-744 transfection inhibited endogenous TGF-β1 synthesis, while direct targeting of TGF-β1 was shown in separate experiments, in which miR-744 decreased TGF-β1 3'UTR reporter activity. This work identifies miR-744-directed post-transcriptional regulation of TGF-β1 which, given the pleiotropic nature of cellular responses to TGF-β1, is potentially widely significant.

  12. Post-transcriptional regulation of wnt8a is essential to zebrafish axis development.

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    Wylie, Annika D; Fleming, Jo-Ann G W; Whitener, Amy E; Lekven, Arne C

    2014-02-01

    wnt8a Is essential for normal patterning during vertebrate embryonic development, and either gain or loss-of-function gene dysregulation results in severe axis malformations. The zebrafish wnt8a locus is structured such that transcripts may possess two regulatory 3' untranslated regions (UTRs), raising the possibility of post-transcriptional regulation as an important mode of wnt8a signaling control. To determine whether both UTRs contribute to post-transcriptional wnt8a gene regulation, each UTR (UTR1 and UTR2) was tested in transient and transgenic reporter assays. Both UTRs suppress EGFP reporter expression in cis, with UTR2 exhibiting a more pronounced effect. UTR2 contains a 6 base sequence necessary for UTR2 regulatory function that is complementary to the seed of the microRNA, miR-430. A target protector morpholino that overlaps the seed complement stabilizes both reporter mRNAs and wnt8a mRNAs, and produces phenotypic abnormalities consistent with wnt8a gain-of-function. In rescue assays, specific functions can be attributed to each of the two wnt8a proteins encoded by the locus. An interplay of wnt8a.1 and wnt8a.2 regulates neural and mesodermal patterning and morphogenesis as well as patterning between brain subdivisions. Thus, post-transcriptional control of wnt8a is essential to fine tune the balance of the signaling outputs of the complex wnt8a locus.

  13. Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.

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    Ahamad, Jamaluddin; Ojha, Sandeep; Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha

    2015-06-01

    Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA accumulates during serum starvation. To investigate the regulation of RP genes under stress we measured transcription of six selected RP genes from the small- and large-ribosomal subunits (RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late-stages of ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during serum starvation and upon serum replenishment. Although luciferase transcript levels remained unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control cells, respectively. After serum replenishment the activity increased to normal levels, suggesting post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation. Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not decline during serum starvation, showing that this sequence is required to repress translation of RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the translation of RP mRNAs on the other.

  14. Post-transcriptional gene regulation by RNA-binding proteins in vascular endothelial dysfunction.

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    Xin, HongBo; Deng, KeYu; Fu, MinGui

    2014-08-01

    Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative and migratory capacity of endothelial cells, as well as control of leukocyte trafficking. Endothelial dysfunction is an early step in vascular inflammatory diseases such as atherosclerosis, diabetic vascular complications, sepsis-induced or severe virus infection-induced organ injuries. The expressions of inflammatory cytokines and vascular adhesion molecules induced by various stimuli, such as modified lipids, smoking, advanced glycation end products and bacteria toxin, significantly contribute to the development of endothelial dysfunction. The transcriptional regulation of inflammatory cytokines and vascular adhesion molecules has been well-studied. However, the regulation of those gene expressions at post-transcriptional level is emerging. RNA-binding proteins have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level in microRNA-dependent or independent manners. This review summarizes the latest insights into the roles of RNA-binding proteins in controlling vascular endothelial cell functions and their contribution to the pathogenesis of vascular inflammatory diseases.

  15. A role for RNA post-transcriptional regulation in satellite cell activation

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    Farina Nicholas H

    2012-10-01

    Full Text Available Abstract Background Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretm and Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors. Results An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate. Conclusions The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation.

  16. An extended gene protein/products boolean network model including post-transcriptional regulation

    Science.gov (United States)

    2014-01-01

    Background Networks Biology allows the study of complex interactions between biological systems using formal, well structured, and computationally friendly models. Several different network models can be created, depending on the type of interactions that need to be investigated. Gene Regulatory Networks (GRN) are an effective model commonly used to study the complex regulatory mechanisms of a cell. Unfortunately, given their intrinsic complexity and non discrete nature, the computational study of realistic-sized complex GRNs requires some abstractions. Boolean Networks (BNs), for example, are a reliable model that can be used to represent networks where the possible state of a node is a boolean value (0 or 1). Despite this strong simplification, BNs have been used to study both structural and dynamic properties of real as well as randomly generated GRNs. Results In this paper we show how it is possible to include the post-transcriptional regulation mechanism (a key process mediated by small non-coding RNA molecules like the miRNAs) into the BN model of a GRN. The enhanced BN model is implemented in a software toolkit (EBNT) that allows to analyze boolean GRNs from both a structural and a dynamic point of view. The open-source toolkit is compatible with available visualization tools like Cytoscape and allows to run detailed analysis of the network topology as well as of its attractors, trajectories, and state-space. In the paper, a small GRN built around the mTOR gene is used to demonstrate the main capabilities of the toolkit. Conclusions The extended model proposed in this paper opens new opportunities in the study of gene regulation. Several of the successful researches done with the support of BN to understand high-level characteristics of regulatory networks, can now be improved to better understand the role of post-transcriptional regulation for example as a network-wide noise-reduction or stabilization mechanisms. PMID:25080304

  17. Insights into post-transcriptional regulation during legume-rhizobia symbiosis.

    Science.gov (United States)

    Reynoso, Mauricio Alberto; Blanco, Flavio Antonio; Zanetti, María Eugenia

    2013-02-01

    During the past ten years, changes in the transcriptome have been assessed at different stages of the legume-rhizobia association by the use of DNA microarrays and, more recently, by RNA sequencing technologies. These studies allowed the identification of hundred or thousand of genes whose steady-state mRNA levels increase or decrease upon bacterial infection or in nodules as compared with uninfected roots. However, transcriptome based-approaches do not distinguish between mRNAs that are being actively translated, stored as messenger ribonucleoproteins (mRNPs) or targeted for degradation. Despite that the increase in steady-state levels of an mRNA does not necessarily correlate with an increase in abundance or activity of the encoded protein, this information has been commonly used to select genes that are candidates to play a role during nodule organogenesis or bacterial infection. Such criterion does not take into account the post-transcriptional mechanisms that contribute to the regulation of gene expression. One of such mechanisms, which has significant impact on gene expression, is the selective recruitment of mRNAs to the translational machinery.  Here, we review the post-transcriptional mechanisms that contribute to the regulation of gene expression in the context of the ecological and agronomical important symbiotic interaction established between roots of legumes and the nitrogen fixing bacteria collectively known as rhizobia. In addition, we discuss how the development of new technologies that allow the assessment of these regulatory layers would help to understand the genetic network governing legume rhizobia symbiosis.

  18. Transcriptional, post-transcriptional and post-translational regulations of gene expression during leaf polarity formation

    Institute of Scientific and Technical Information of China (English)

    Lin Xu; Li Yang; Hai Huang

    2007-01-01

    Leaf morphogenesis requires the establishment of adaxial-abaxial polarity after primordium initiation from the shoot apical meristem (SAM). Several families of transcription factors are known to play critical roles in promoting adaxial or abaxial leaf fate. Recently, post-transcriptional gene silencing pathways have been shown to regulate the establishment of leaf polarity, providing novel and exciting insights into leaf development. For example, microRNAs (miR165/166)and a trans-acting siRNA (TAS3-derived tasiR-ARF) have been shown to repress the expression of several key transcription factor genes. In addition, yet another level of regulation, post-translational regulation, has been revealed recently by studies on the role of the 26S proteasome in leaf polarity. Although our understanding regarding the molecular mechanisms underlying establishment of adaxial-abaxial polarity has greatly improved, there is still much that remains elusive.This review aims to discuss recent progress, as well as the remaining questions, regarding the molecular mechanisms underlying leaf polarity formation.

  19. POST-TRANSCRIPTIONAL REGULATION OF P21WAF1/CIP1 BY P53

    Institute of Scientific and Technical Information of China (English)

    季加孚; 张霁; 焦春雨; 顾晋; 谭立新; 张平; 李培详

    2001-01-01

    Objective: To investigate the post-transcriptional regulation of p21WAF1/CIP1 by p53. Methods: The MDA-MB-468 cells have endogenous mutant p53 and the MCF7 cells lines have wtp53. Recombinant p53 expression and p21WAF1/CIP1 induction were detected by Western blot analysis. Northern blot analysis was carried out to examine whether changes in p21WAF1/CIP1 protein levels in MCF7 cells treated with AdCMVp53 are reflected at the mRNA level. Flow cytometric analysis of MCF7 cells following overexpression of recombination. Results: The ratio of p53: p21WAF1/CIP1 was below 1 at the early stages of AdCMVp53 infection, but increased to 1.6 by day 3 and to 9.7 by day 5 post-infection. As expected, p21WAF1/CIP1 expression was not detectable in MDA-MB-468 cells despite the presence of high levels of mutant p53 protein. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. By Northern blot analyzing the p21WAF1/CIP1: GAPDH ratios at different time points against the ratio at time point 0, a maximum 3-fold induction of p21WAF1/CIP1 mRNA expression relative to untreated control was observed on day 1 post-infection. The flow cytometric analysis indicated that MCF7 cells infected with AdCMVp53 undergo G1 arrest at both time points studied, with G1/S ratios ranging from 5.54 at day 1 to 5.65 at day 7. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. Conclusion: This studydemonstrated that p53 could regulate p21WAF1/CIP1 gene expression at both the transcriptional and post-transcriptional levels in MCF7 cells. The latter mechanism may be involved in or be responsible for, the induction of cell cycle arrest by transcription-defective mutants of p53.

  20. Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots.

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    D'haeseleer, Katrien; Den Herder, Griet; Laffont, Carole; Plet, Julie; Mortier, Virginie; Lelandais-Brière, Christine; De Bodt, Stefanie; De Keyser, Annick; Crespi, Martin; Holsters, Marcelle; Frugier, Florian; Goormachtig, Sofie

    2011-08-01

    • Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

  1. Post-transcriptional regulation of gene expression in neural stem cells.

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    Kim, Do-Yeon

    2016-06-01

    Expression of each gene can be controlled at several steps during the flow of genetic information from DNA to protein. Tight regulation of gene expression is especially important for stem cells because of their greater ripple effects, compared with terminally differentiated cells. Dysregulation of gene expression arising in stem cells can be perpetuated within the stem cell pool via self-renewal throughout life. In addition, transcript profiles within stem cells can determine the selective advantage or disadvantage of each cell, leading to changes in cell fate, such as a tendency for proliferation, death, and differentiation. The identification of neural stem/progenitor cells (NSPCs) and greater understanding of their cellular physiology have raised the possibility of using NSPCs to replace damaged or injured neurons. However, an accurate grasp of gene expression control must take precedence in order to use NSPCs in therapies for neurological diseases. Recently, accumulating evidence has demonstrated the importance of post-transcriptional regulation in NSPC fate decisions. In this review, we will summarize and discuss the recent findings on key mRNA modulators and their vital roles in NSPC homeostasis. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Dysregulation of microRNA-219 promotes neurodegeneration through post-transcriptional regulation of tau

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    Santa-Maria, Ismael; Alaniz, Maria E.; Renwick, Neil; Cela, Carolina; Fulga, Tudor A.; Van Vactor, David; Tuschl, Thomas; Clark, Lorraine N.; Shelanski, Michael L.; McCabe, Brian D.; Crary, John F.

    2015-01-01

    Tau is a highly abundant and multifunctional brain protein that accumulates in neurofibrillary tangles (NFTs), most commonly in Alzheimer’s disease (AD) and primary age-related tauopathy. Recently, microRNAs (miRNAs) have been linked to neurodegeneration; however, it is not clear whether miRNA dysregulation contributes to tau neurotoxicity. Here, we determined that the highly conserved brain miRNA miR-219 is downregulated in brain tissue taken at autopsy from patients with AD and from those with severe primary age-related tauopathy. In a Drosophila model that produces human tau, reduction of miR-219 exacerbated tau toxicity, while overexpression of miR-219 partially abrogated toxic effects. Moreover, we observed a bidirectional modulation of tau levels in the Drosophila model that was dependent on miR-219 expression or neutralization, demonstrating that miR-219 regulates tau in vivo. In mammalian cellular models, we found that miR-219 binds directly to the 3′-UTR of the tau mRNA and represses tau synthesis at the post-transcriptional level. Together, our data indicate that silencing of tau by miR-219 is an ancient regulatory mechanism that may become perturbed during neurofibrillary degeneration and suggest that this regulatory pathway may be useful for developing therapeutics for tauopathies. PMID:25574843

  3. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

    2015-06-15

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

  4. Implication of CA repeated tracts on post-transcriptional regulation in Trypanosoma cruzi.

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    Pastro, Lucía; Smircich, Pablo; Pérez-Díaz, Leticia; Duhagon, María Ana; Garat, Beatriz

    2013-08-01

    In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.

  5. Post-transcriptional regulator Hfq binds catalase HPII: crystal structure of the complex.

    Directory of Open Access Journals (Sweden)

    Koji Yonekura

    Full Text Available We report a crystal structure of Hfq and catalase HPII from Escherichia coli. The post-transcriptional regulator Hfq plays a key role in the survival of bacteria under stress. A small non-coding RNA (sRNA DsrA is required for translation of the stationary phase sigma factor RpoS, which is the central regulator of the general stress response. Hfq facilitates efficient translation of rpoS mRNA, which encodes RpoS. Hfq helps in the function of other specific proteins involved in RNA processing, indicating its versatility in the cell. However, structural information regarding its interactions with partners is missing. Here we obtained crystals of Hfq and HPII complexes from cell lysates following attempts to overexpress a foreign membrane protein. HPII is one of two catalases in E. coli and its mRNA is transcribed by an RNA polymerase holoenzyme containing RpoS, which in turn is under positive control of small non-coding RNAs and of the RNA chaperone Hfq. This sigma factor is known to have a pronounced effect on the expression of HPII. The crystal structure reveals that a Hfq hexamer binds each subunit of a HPII tetramer. Each subunit of the Hfq hexamer exhibits a unique binding mode with HPII. The hexamer of Hfq interacts via its distal surface. The proximal and distal surfaces are known to specifically bind different sRNAs, and binding of HPII could affect Hfq function. Hfq-HPII complexation has no effect on catalase HPII activity.

  6. The post-transcriptional operon

    DEFF Research Database (Denmark)

    Tenenbaum, Scott A.; Christiansen, Jan; Nielsen, Henrik

    2011-01-01

    A post-transcriptional operon is a set of monocistronic mRNAs encoding functionally related proteins that are co-regulated by a group of RNA-binding proteins and/or small non-coding RNAs so that protein expression is coordinated at the post-transcriptional level. The post-transcriptional operon...... model (PTO) is used to describe data from an assortment of methods (e.g. RIP-Chip, CLIP-Chip, miRNA profiling, ribosome profiling) that globally address the functionality of mRNA. Several examples of post-transcriptional operons have been documented in the literature and demonstrate the usefulness...

  7. 3{prime} UTR sequence-specific mRNA-protein complexes and the post-transcriptional regulation of catalase

    Energy Technology Data Exchange (ETDEWEB)

    Reimer, D.L.; Ott, R.N.; Singh, S.M. [Univ. of Western Ontario, London (Canada)

    1994-09-01

    Recently, sequences in the 3{prime} untranslated region (3{prime} UTR) of some genes have been recognized which may play an important role in the post-transcriptional regulation of gene expression. Mutations in this region of the gene are known to cause at least two diseases including myotonic dystrophy and a lysosomal accumulation disease. The mechanism is thought to involve mRNA-protein interactions that affect translation and/or mRNA stability. Reports of this nature are not common and the significance of the often large 3{prime} UTR on the regulation of gene expression remains speculative. Studies on the 3{prime} UTR mRNA-protein interactions in model eukaryotic genes therefore are critical to better understand the molecular mechanisms involved in post-transcriptional gene regulation. Mouse catalase, encoded by Cas-1, was used as a model to characterize the molecular mechanisms of post-transcriptional gene regulation. The 3{prime} UTR (752 bp) of Cas-1 contains three unusual, near repeats [(CA){sub 31}, (U){sub 15} and (TGTGC){sub 7}]. Gel mobility shift assays using {sup 32}P-labelled transcripts which contain these sequences and tissue homogenates from various sources identified mRNA-protein complexes specific to (CA){sub 31} and (U){sub 15} only. In all strains analyzed, a single protein of 69 kDa which was involved in the (CA){sub 31} complex, was observed in most tissues except lung and was localized to the polysomal fraction. Similarly, two proteins involved in the (U){sub 15} complex, 38 and 47 kDa, were observed in all tissues and strains studied. Only the 38 kDa protein was observed in the polysomal fraction. The results argue for a possible role for these 3{prime} UTR mRNA-binding protein complexes in the post-transcriptional regulation of this antioxidant enzyme.

  8. Caveolin-1-Mediated Expression and Secretion of Kallikrein 6 in Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Rebecca S. Henkhaus

    2008-02-01

    Full Text Available Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6 has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1, the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion.

  9. Matrin 3 is a co-factor for HIV-1 Rev in regulating post-transcriptional viral gene expression

    Directory of Open Access Journals (Sweden)

    Jeang Kuan-Teh

    2011-07-01

    Full Text Available Abstract Post-transcriptional regulation of HIV-1 gene expression is mediated by interactions between viral transcripts and viral/cellular proteins. For HIV-1, post-transcriptional nuclear control allows for the export of intron-containing RNAs which are normally retained in the nucleus. Specific signals on the viral RNAs, such as instability sequences (INS and Rev responsive element (RRE, are binding sites for viral and cellular factors that serve to regulate RNA-export. The HIV-1 encoded viral Rev protein binds to the RRE found on unspliced and incompletely spliced viral RNAs. Binding by Rev directs the export of these RNAs from the nucleus to the cytoplasm. Previously, Rev co-factors have been found to include cellular factors such as CRM1, DDX3, PIMT and others. In this work, the nuclear matrix protein Matrin 3 is shown to bind Rev/RRE-containing viral RNA. This binding interaction stabilizes unspliced and partially spliced HIV-1 transcripts leading to increased cytoplasmic expression of these viral RNAs.

  10. The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

    Science.gov (United States)

    Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

    2016-01-01

    Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27kip1 and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27kip1 mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. PMID:26926106

  11. Up-regulation of thromboxane A2 receptor expression by lipid soluble smoking particles through post-transcriptional mechanisms

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2008-01-01

    . The present study was designed to test if lipid soluble smoking particles (DSP) enhance TxA(2) receptor (TP) expression in rat mesenteric arteries, and if intracellular mitogen-activated protein kinase (MAPK) pathways play a role. Organ culture of rat mesenteric arteries in the presence of DSP (0.2 microl...... actinomycin D, but was almost completely abolished by cycloheximide, a general translational inhibitor. Dexamethasone, a glucocorticoid, manifested a potent inhibitory effect as well. These results suggest that the up-regulation of TP receptor occurs via post-transcriptional events, and mainly translation...... are responsible for the up-regulation of TP receptor by DSP, in which enhanced translation is the major cause of the elevated protein expression and the enhanced contraction....

  12. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    Energy Technology Data Exchange (ETDEWEB)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  13. Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection*

    Science.gov (United States)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen. PMID:23970565

  14. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection.

    Science.gov (United States)

    Nakayasu, Ernesto S; Tempel, Rebecca; Cambronne, Xiaolu A; Petyuk, Vladislav A; Jones, Marcus B; Gritsenko, Marina A; Monroe, Matthew E; Yang, Feng; Smith, Richard D; Adkins, Joshua N; Heffron, Fred

    2013-11-01

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen.

  15. Post-transcriptional regulation of dopamine D1 receptor expression in caudate-putamen of cocaine-sensitized mice.

    Science.gov (United States)

    Tobón, Krishna E; Catuzzi, Jennifer E; Cote, Samantha R; Sonaike, Adenike; Kuzhikandathil, Eldo V

    2015-07-01

    The dopamine D1 receptor is centrally involved in mediating the effects of cocaine and is essential for cocaine-induced locomotor sensitization. Changes in D1 receptor expression have been reported in various models of cocaine addiction; however, the mechanisms that mediate these changes in D1 receptor expression are not well understood. Using preadolescent drd1a-EGFP mice and a binge cocaine treatment protocol we demonstrate that the D1 receptor is post-transcriptionally regulated in the caudate-putamen of cocaine-sensitized animal. While cocaine-sensitized mice express high levels of steady-state D1 receptor mRNA, the expression of D1 receptor protein is not elevated. We determined that the post-transcriptional regulation of D1 receptor mRNA is rapidly attenuated and D1 receptor protein levels increase within 30 min when the sensitized mice are challenged with cocaine. The rapid increase in D1 receptor protein levels requires de novo protein synthesis and correlates with the cocaine-induced hyperlocomotor activity in the cocaine-sensitized mice. The increase in D1 receptor protein levels in the caudate-putamen inversely correlated with the levels of microRNA 142-3p and 382, both of which regulate D1 receptor protein expression. The levels of these two microRNAs decreased significantly within 5 min of cocaine challenge in sensitized mice. The results provide novel insights into the previously unknown rapid kinetics of D1 receptor protein expression which occurs in a time scale that is comparable to the expression of immediate early genes. Furthermore, the results suggest a potential novel role for inherently labile microRNAs in regulating the rapid expression of D1 receptor protein in cocaine-sensitized animals.

  16. Hypermethylation and post-transcriptional regulation of DNA methyltransferases in the ovarian carcinomas of the laying hen.

    Science.gov (United States)

    Lee, Jin-Young; Jeong, Wooyoung; Lim, Whasun; Lim, Chul-Hong; Bae, Seung-Min; Kim, Jinyoung; Bazer, Fuller W; Song, Gwonhwa

    2013-01-01

    DNA methyltransferases (DNMTs) are key regulators of DNA methylation and have crucial roles in carcinogenesis, embryogenesis and epigenetic modification. In general, DNMT1 has enzymatic activity affecting maintenance of DNA methylation, whereas DNMT3A and DNMT3B are involved in de novo methylation events. Although DNMT genes are well known in mammals including humans and mice, they are not well studied in avian species, especially the laying hen which is recognized as an excellent animal model for research on human ovarian carcinogenesis. Results of the present study demonstrated that expression of DNMT1, DNMT3A and DNMT3B genes was significantly increased, particularly in the glandular epithelia (GE) of cancerous ovaries, but not normal ovaries. Consistent with this result, immunoreactive 5-methylcytosine protein was predominantly abundant in nuclei of stromal and GE cells of cancerous ovaries, but it was also found that, to a lesser extent, in nuclei of stromal cells of normal ovaries. Methylation-specific PCR analysis detected hypermethylation of the promoter regions of the tumor suppressor genes in the initiation and development of chicken ovarian cancer. Further, several microRNAs, specifically miR-1741, miR-16c, and miR-222, and miR-1632 were discovered to influence expression of DNMT3A and DNMT3B, respectively, via their 3'-UTR which suggests post-transcriptional regulation of their expression in laying hens. Collectively, results of the present study demonstrated increased expression of DNMT genes in cancerous ovaries of laying hens and post-transcriptional regulation of those genes by specific microRNAs, as well as control of hypermethylation of the promoters of tumor suppressor genes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    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 its 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.

  18. Post-transcriptional regulation of NifA expression by Hfq and RNase E complex in Rhizobium leguminosarum bv. viciae

    Institute of Scientific and Technical Information of China (English)

    Yinghua Zhang; Guofan Hong

    2009-01-01

    NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum by. viciae strain 8401/pRL1JI, the NifA gene is part of a gene cluster (fixABCXNifAB). In this study, results showed that in R. leguminosarum by. viciae 8401/pRLI1I, host factor required (Hfq), and RNase E were involved in the post-transcriptional regulation of NifA expression. It was found that Hfq-dependent RNase E cleavage of NifA mRNA was essen-tial for NifA translation. The cleavage site is located at 32 nucleotides upstream of the NifA translational start codon. A possible explanation based on predicted RNA secondary structure of the NifA 5'-untranslated region was that the cleavage made ribosome-binding sites accessible for translation.

  19. Transcriptional and post-transcriptional regulation of SPAST, the gene most frequently mutated in hereditary spastic paraplegia.

    Directory of Open Access Journals (Sweden)

    Brian J Henson

    Full Text Available Hereditary spastic paraplegias (HSPs comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70-80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3'-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP.

  20. Effect of BRAFV600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model

    Directory of Open Access Journals (Sweden)

    Guenther Simone M

    2007-03-01

    Full Text Available Abstract Background microRNAs (miRNAs are a group of non-coding single stranded RNAs measuring approximately 22 nucleotides in length that have been found to control cell growth, differentiation and apoptosis. They negatively regulate target genes and have recently been implicated in tumourigenesis. Furthermore, miRNA expression profiling correlates with various cancers, with these genes thought to act as both tumour suppressors and oncogenes. Recently, a point mutation in the BRAF gene leading to a V600E substitution has been identified as the most common genetic change in papillary thyroid carcinoma (PTC occurring in 29–69% of cases. This mutation leads to aberrant MAPK activation that is implicated in tumourigenesis. Aim The aim of this study was to identify the effect that BRAF oncogene has on post-transcriptional regulation in PTC by using microRNA analysis. Results A unique miRNA expression signature differentiated between PTC cell lines with BRAF mutations and a normal thyroid cell line. 15 miRNAs were found to be upregulated and 23 miRNAs were downregulated. Several of these up/down regulated miRNAs may be involved in PTC pathogenesis. miRNA profiling will assist in the elucidation of disease pathogenesis and identification biomarkers and targets.

  1. Impaired Post-transcriptional Regulation in Alzheimer's Disease%转录后基因调控异常与老年性痴呆

    Institute of Scientific and Technical Information of China (English)

    朱笠; 刘江红; 吴瑛

    2012-01-01

    Post-transcriptional regulation plays a central role in the development and maintenance of the nervous system. Defects in post-transcriptional regulation lead to neurodegenerative diseases. Mounting evidence suggests that the impaired post-transcriptional regulation of associated genes contribute to the neurodegenerative process, such as Alzheimer's disease (AD). This review discusses the role of disruption of alternative splicing regulation of the human tau gene (MAPT) and alterations of microRNA expression in the pathogenesis of neurodegeneration including AD. A brief overview is provided for the role and the mechanism of post-transcriptional regulation in AD pathogenesis.%转录后基因调控异常与阿尔茨海默病(Alzheimer's disease,AD)发生发展的关系研究越来越受到重视.本文重点论述了tau基因(MAPT)发生可变剪接异常与AD发生的关系,以及参与转录后调控的RNA结合蛋白和非编码RNA在AD发生发展中的作用.

  2. Post-transcriptional regulation of ethylene perception and signaling in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Schaller, George Eric

    2014-03-19

    The simple gas ethylene functions as an endogenous regulator of plant growth and development, and modulates such energy relevant processes as photosynthesis and biomass accumulation. Ethylene is perceived in the plant Arabidopsis by a five-member family of receptors related to bacterial histidine kinases. Our data support a general model in which the receptors exist as parts of larger protein complexes. Our goals have been to (1) characterize physical interactions among members of the signaling complex; (2) the role of histidine-kinase transphosphorylation in signaling by the complex; and (3) the role of a novel family of proteins that regulate signal output by the receptors.

  3. Post-Transcriptional Regulation by the Csr Global Regulatory System in Escherichia coli

    OpenAIRE

    Suzuki, Kazushi; 鈴木, 一史

    2007-01-01

    In many species of bacteria, the Csr (carbon storage regulator) global regulatory system coordinates the expression of various genes. In Escherichia coli, the central component of this system, CsrA, is a RNA-binding protein. The CsrA is a homodimer and binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two small non-coding RNAs, CsrB and CsrC. These RNAs contain multiple CsrA-binding sequences and act by sequestering CsrA. In thi...

  4. Post-transcriptional regulation of gene PA5507 controls PQS concentration in Pseudomonas aeruginosa

    OpenAIRE

    Tipton, Kyle A.; Coleman, James P.; Pesci, Everett C.

    2015-01-01

    Pseudomonas aeruginosa can sense and respond to a myriad of environmental signals and utilizes a system of small molecules to communicate through intercellular signaling. The small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal [PQS]) is one of these signals and its synthesis is important for virulence. Previously, we identified an RpiR-type transcriptional regulator, QapR, that positively affects PQS production by repressing the qapR operon. An in-frame deletion of thi...

  5. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    Science.gov (United States)

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B

    1990-11-01

    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  6. Post-transcriptional regulation of neurofibromin level in cultured human melanocytes in response to growth factors.

    Science.gov (United States)

    Griesser, J; Kaufmann, D; Maier, B; Mailhammer, R; Kuehl, P; Krone, W

    1997-03-01

    Among the symptoms that characterize neurofibromatosis type 1 (NF1) are pigmentation anomalies such as cafe au lait spots. It has been suggested that the reduction of the neurofibromin level in the epidermis of NF1 patients is responsible for the observed signs such as altered melanogenesis and altered density of melanocytes. Our studies show that in cultured normal human melanocytes, the neurofibromin level can be varied in vitro over a wide range by using different culture conditions. The influence of factors that control differentiation and proliferation of melanocytes on neurofibromin levels was studied. Immunoprecipitation followed by western blotting showed a 3- to 4-fold increase of neurofibromin after stimulation by PMA or bFGF, respectively, and a 1.5-fold increase in cells stimulated with steel factor. The increase of neurofibromin was not paralleled by a higher NF1 mRNA level as proved by northern blotting. Pulse-chase experiments with 35S-labeled melanocytes revealed an approximately 3-fold increase in the half-life of neurofibromin in bFGF- or PMA-stimulated cells compared to controls. These results indicate that the neurofibromin level of cultured melanocytes can be regulated by a mechanism independent of NF1 gene transcription and translation, which might influence the degradation rate of the protein.

  7. Post-transcriptional regulation on a global scale: form and function of Csr/Rsm systems.

    Science.gov (United States)

    Romeo, Tony; Vakulskas, Christopher A; Babitzke, Paul

    2013-02-01

    Originally described as a repressor of gene expression in the stationary phase of growth, CsrA (RsmA) regulates primary and secondary metabolic pathways, biofilm formation, motility, virulence circuitry of pathogens, quorum sensing and stress response systems by binding to conserved sequences in its target mRNAs and altering their translation and/or turnover. While the binding of CsrA to RNA is understood at an atomic level, new mechanisms of gene activation and repression by this protein are still emerging. In the γ-proteobacteria, small non-coding RNAs (sRNAs) use molecular mimicry to sequester multiple CsrA dimers away from mRNA. In contrast, the FliW protein of Bacillus subtilis inhibits CsrA activity by binding to this protein, thereby establishing a checkpoint in flagellum morphogenesis. Turnover of CsrB and CsrC sRNAs in Escherichia coli requires a specificity protein of the GGDEF-EAL domain superfamily, CsrD, in addition to the housekeeping nucleases RNase E and PNPase. The Csr system of E. coli contains extensive autoregulatory circuitry, which governs the expression and activity of CsrA. Interaction of the Csr system with transcriptional regulatory networks results in a variety of complex response patterns. This minireview will highlight basic principles and new insights into the workings of these complex eubacterial regulatory systems.

  8. RNA-Seq analysis of rice roots reveals the involvement of post-transcriptional regulation in response to cadmium stress

    Directory of Open Access Journals (Sweden)

    Luqing eZheng

    2015-12-01

    Full Text Available Widely-spread cadmium (Cd pollution in the soil threatens both crop production and human health. How plants deal with the excess Cd are largely unknown. To evaluate the molecular mechanism by which plants respond to Cd stress, rice seedlings were treated with two concentrations of Cd and subjected to deep RNA sequencing. Comprehensive RNA-Seq analysis of rice roots under two gradients of Cd treatment revealed 1,169 Cd toxicity-responsive genes. These genes were involved in the reactive oxygen species scavenging system, stress response, cell wall formation, ion transport, and signal transduction. Nine out of 93 predicted long non coding RNAs (lncRNAs were detected as Cd-responsive lncRNAs due to their high correlation with the Cd stress response. In addition, we analyzed alternative splicing (AS events under different Cd concentrations. 476 differential alternatively spliced genes with 542 aberrant splicing events were identified. GO analysis indicated that these genes were highly enriched in oxidation reduction and cellular response to chemical stimulus. Real-time qRT-PCR validation analysis strengthened the reliability of our RNA-Seq results. The results suggest that post-transcriptional AS regulation may also be involved in plant responses to high Cd stress.

  9. Modulation of Sinorhizobium meliloti quorum sensing by Hfq-mediated post-transcriptional regulation of ExpR.

    Science.gov (United States)

    Gao, Mengsheng; Tang, Ming; Guerich, Lois; Salas-Gonzalez, Isai; Teplitski, Max

    2015-02-01

    In Sinorhizobium meliloti, the timing of quorum sensing (QS)-dependent gene expression is controlled at multiple levels. RNA binding protein Hfq contributes to the regulation of QS signal production, and this regulation is exerted both in the manner that involves the acyl homoserine lactone receptor ExpR, and via expR-independent mechanisms. In the expR+ strain of S. meliloti, deletion of hfq resulted in the hyper-accumulation of QS signals at low population densities, increased diversity of the QS signals in mid-to-late exponential phase and then led to a sharp decrease in QS signal accumulation in stationary phase. Quantitative polymerase chain reaction revealed that the accumulation of expR and sinI (but not sinR) mRNA was increased in the late exponential phase in an hfq-dependent manner. A translational, but not transcriptional, expR-uidA reporter was controlled by hfq, while both transcriptional and translational sinI-uidA reporters were regulated in the hfq-dependent manner. In co-immunoprecipation experiments, expR mRNA was bound to and then released from Hfq, similar to the positive controls (small regulatory RNA SmrC9, SmrC15, SmrC16 and SmrC45). Neither sinI nor sinR transcripts were detected in the pool of RNA heat-released from Hfq-RNA complexes. Therefore, post-transcriptional regulator Hfq controls the production and perception of QS signals, and at higher population densities this control is mediated directly via interactions with expR.

  10. Isoforms of elongation factor eEF1A may be differently regulated at post-transcriptional level in breast cancer progression

    Directory of Open Access Journals (Sweden)

    Vislovukh A. A.

    2013-01-01

    Full Text Available Eukaryotic translation elongation factor 1A exists as two 98 % homologous isoforms: eEF1A1 (A1 and eEF1A2 (A2 which are tissue and development specific. Despite high homology in an open reading frame (ORF region, mRNAs coding for eEF1A1 and eEF1A2 are different in their untranslated regions (UTR, suggesting a possibility of their dissimilar post-transcriptional regulation. Aim. To analyze the existence of cis-acting motifs in the UTRs of EEF1A1/A2 mRNAs, to confirm the possibility of post-transcriptional control of eEF1A1 and eEF1A2 expression. Methods. An ensemble of bioinformatic methods was applied to predict regulatory motifs in the UTRs of EEF1A1/A2 mRNAs. Dual-luciferase reporter assay was employed to detect post-transcriptional regulation of eEF1A1/A2 expression. Results. Numerous regulatory motifs in the UTR of EEF1A1/A2 mRNAs were found bioinformatically. The experimental evidence was obtained for the existence of negative regulation of EEF1A1 and positive regulation of EEF1A2 mRNA in the model of breast cancer development. Conclusions. EEF1A1 and EEF1A2 mRNAs contain distinct motifs in the UTRs and are differently regulated in cancer suggesting the possibility of their control by different cellular signals.

  11. Role of miR-142-3p in the post-transcriptional regulation of the clock gene Bmal1 in the mouse SCN.

    Science.gov (United States)

    Shende, Vikram R; Neuendorff, Nichole; Earnest, David J

    2013-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional modulators by regulating stability or translation of target mRNAs. Recent studies have implicated miRNAs in the regulation of mammalian circadian rhythms. To explore the role of miRNAs in the post-transcriptional modulation of core clock genes in the master circadian pacemaker, we examined miR-142-3p for evidence of circadian expression in the suprachiasmatic nuclei (SCN), regulation of its putative clock gene target Bmal1 via specific binding sites in the 3' UTR and overexpression-induced changes in the circadian rhythm of BMAL1 protein levels in SCN cells. In mice exposed to constant darkness (DD), miR-142-3p levels in the SCN were characterized by circadian rhythmicity with peak expression during early subjective day at CT 3. Mutagenesis studies indicate that two independent miRNA recognition elements located at nucleotides 1-7 and 335-357 contribute equally to miR-142-3p-induced repression of luciferase-reported Bmal1 3' UTR activity. Importantly, overexpression of miR-142-3p in immortalized SCN cells abolished circadian variation in endogenous BMAL1 protein levels in vitro. Collectively, our results suggest that miR-142-3p may play a role in the post-transcriptional modulation of Bmal1 and its oscillatory regulation in molecular feedback loops mediating SCN circadian function.

  12. Role of miR-142-3p in the post-transcriptional regulation of the clock gene Bmal1 in the mouse SCN.

    Directory of Open Access Journals (Sweden)

    Vikram R Shende

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that function as post-transcriptional modulators by regulating stability or translation of target mRNAs. Recent studies have implicated miRNAs in the regulation of mammalian circadian rhythms. To explore the role of miRNAs in the post-transcriptional modulation of core clock genes in the master circadian pacemaker, we examined miR-142-3p for evidence of circadian expression in the suprachiasmatic nuclei (SCN, regulation of its putative clock gene target Bmal1 via specific binding sites in the 3' UTR and overexpression-induced changes in the circadian rhythm of BMAL1 protein levels in SCN cells. In mice exposed to constant darkness (DD, miR-142-3p levels in the SCN were characterized by circadian rhythmicity with peak expression during early subjective day at CT 3. Mutagenesis studies indicate that two independent miRNA recognition elements located at nucleotides 1-7 and 335-357 contribute equally to miR-142-3p-induced repression of luciferase-reported Bmal1 3' UTR activity. Importantly, overexpression of miR-142-3p in immortalized SCN cells abolished circadian variation in endogenous BMAL1 protein levels in vitro. Collectively, our results suggest that miR-142-3p may play a role in the post-transcriptional modulation of Bmal1 and its oscillatory regulation in molecular feedback loops mediating SCN circadian function.

  13. Post-transcriptional regulation of the human reduced folate carrier as a novel adaptive mechanism in response to folate excess or deficiency

    OpenAIRE

    Hou, Zhanjun; Orr, Steve; Matherly, Larry H.

    2014-01-01

    The RFC (reduced folate carrier) is the principal mechanism by which folates and clinically used antifolates are delivered to mammalian cells. hRFC (human RFC) is subject to complex transcriptional controls and exists as homo-oligomer. To explore the post-transcriptional regulation of hRFC by exogenous folates, hRFC-null HeLa cells were stably transfected with hRFC under control of a constitutive promoter. hRFC transcripts and the total membrane protein increased with increasing LCV [(6R,S)5-...

  14. Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell lines.

    Science.gov (United States)

    Mei, Chuanzhong; Sun, Lidong; Liu, Yonglei; Yang, Yong; Cai, Xiumei; Liu, Mingzhu; Yao, Wantong; Wang, Can; Li, Xin; Wang, Liying; Li, Zengxia; Shi, Yinghong; Qiu, Shuangjian; Fan, Jia; Zha, Xiliang

    2010-09-01

    DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

  15. Conserved miRNAs are candidate post-transcriptional regulators of developmental arrest in free-living and parasitic nematodes.

    Science.gov (United States)

    Ahmed, Rina; Chang, Zisong; Younis, Abuelhassan Elshazly; Langnick, Claudia; Li, Na; Chen, Wei; Brattig, Norbert; Dieterich, Christoph

    2013-01-01

    Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved "dauer-infective" expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.

  16. RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7.

    Science.gov (United States)

    Cascajo, María V; Abdelmohsen, Kotb; Noh, Ji Heon; Fernández-Ayala, Daniel J M; Willers, Imke M; Brea, Gloria; López-Lluch, Guillermo; Valenzuela-Villatoro, Marina; Cuezva, José M; Gorospe, Myriam; Siendones, Emilio; Navas, Plácido

    2016-07-01

    Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

  17. Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

    Science.gov (United States)

    Lu, Sizhao; Mott, Justin L; Harrison-Findik, Duygu Dee

    2015-10-02

    Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

  18. Responsiveness of Trichomonas vaginalis to iron concentrations: evidence for a post-transcriptional iron regulation by an IRE/IRP-like system.

    Science.gov (United States)

    Torres-Romero, J C; Arroyo, R

    2009-12-01

    Trichomonas vaginalis has high iron-dependency, favoring its growth and multiplication in culture. Iron also regulates some of the trichomonal virulence properties by yet unknown mechanisms. Iron is an essential but potentially toxic metal for the majority of organisms. Thus, its concentration must be tightly regulated within the cell. In mammals, the iron homeostasis is mainly regulated at the post-transcriptional level by a well known mechanism mediated by the binding of iron regulatory proteins (IRP1 and IRP2) to hairpin-loop structures, dubbed iron-responsive elements (IREs), localized in the untranslated regions (UTRs) of target mRNAs. The knowledge of iron regulation in T. vaginalis is still very limited. An iron-responsive promoter and other regulatory elements in the 5'-UTR of the ap65-1 gene were identified as a mechanism for the positive transcriptional regulation of trichomonad genes by iron. Recently, two IRE-like hairpin-loop structures in mRNAs of differentially iron-regulated TVCP4 and TVCP12 cysteine proteinases, as well as IRP-like trichomonad proteins were identified in T. vaginalis, suggesting the existence in this protozoan of a post-transcriptional iron regulatory mechanism by an IRE/IRP-like system. The responsiveness of T. vaginalis to distinct iron concentrations was examined here. Also, the comparison of the atypical IRE-like sequences of T. vaginalis with the consensus IRE and other putative IRE sequences present in parasite and bacteria mRNAs suggest that these trichomonad IRE-like sequences might be the ancestral forms of the RNA stem-loop structures of the IRE/IRP system.

  19. DoRiNA 2.0--upgrading the doRiNA database of RNA interactions in post-transcriptional regulation.

    Science.gov (United States)

    Blin, Kai; Dieterich, Christoph; Wurmus, Ricardo; Rajewsky, Nikolaus; Landthaler, Markus; Akalin, Altuna

    2015-01-01

    The expression of almost all genes in animals is subject to post-transcriptional regulation by RNA binding proteins (RBPs) and microRNAs (miRNAs). The interactions between both RBPs and miRNAs with mRNA can be mapped on a whole-transcriptome level using experimental and computational techniques established in the past years. The combined action of RBPs and miRNAs is thought to form a post-transcriptional regulatory code. Here we present doRiNA 2.0, available at http://dorina.mdc-berlin.de. In this highly improved new version, we have completely reworked the user interface and expanded the database to improve the usability of the website. Taking into account user feedback over the past years, the input forms for both the simple and the combinatorial search function have been streamlined and combined into a single web page that will also display the search results. Especially, custom uploads is one of the key new features in doRiNA 2.0. To enable the inclusion of doRiNA into third-party analysis pipelines, all operations are accessible via a REST API. Alternatively, local installations can be queried using a Python API. Both the web application and the APIs are available under an OSI-approved Open Source license that allows research and commercial access and re-use.

  20. Transcriptional and post-transcriptional regulation of retrotransposons IAP and MuERV-L affect pluripotency of mice ES cells

    Directory of Open Access Journals (Sweden)

    Pintado Belen

    2006-11-01

    Full Text Available Abstract Background In the mouse, culture of embryonic stem (ES cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs. In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it. Methods In order to investigate whether this phenotype was associated with chromosome alteration, inadvertent differentiation, or epigenetic modification, we characterized and compared this R1 ES cell line at passage 27 with an early passage and with a second ES cell line C57/CBAF1 generated in our laboratory. We assessed: i karyotype; ii expression of pluripotent and differentiation markers, iii mRNA transcription by qRT-PCR of two REs, intracisternal-A particle (IAP and murine endogenous-retrovirus-L (MuERV-L, and iv methylation of IAP and MuERV-L. Results The R1 ES cell at passage 27, presented normal morphology, karyotype, and expression of genetic markers characteristic of pluripotent; however, it was detected an altered mRNA transcription of sense and antisense RNA strands of both REs, concomitantly with an altered methylation pattern for the IAP element but not for MuERV-L. These results indicate that besides methylation, other post-transcriptional processes are involved in gene silencing of some REs; and that culture of ES cells may decrease their pluripotency by producing inadvertent alterations in the expression of REs without significantly affecting the morphology, chromosome structure, and expression of pluripotent or differentiation markers. Conclusion Inadvertent REs instability may have important consequences for the use of ES cells in

  1. Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

    Science.gov (United States)

    Rivera Vargas, T; Boudoukha, S; Simon, A; Souidi, M; Cuvellier, S; Pinna, G; Polesskaya, A

    2014-05-29

    RNA-binding proteins of the IMP family (insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1-3) are important post-transcriptional regulators of gene expression. Multiple studies have linked high expression of IMP proteins, and especially of IMP-3, to an unfavorable prognosis in numerous types of cancer. The specific importance of IMP-3 for cancer transformation remains poorly understood. We here show that all three IMPs can directly bind the mRNAs of cyclins D1, D3 and G1 (CCND1, D3 and G1) in vivo and in vitro, and yet only IMP-3 regulates the expression of these cyclins in a significant manner in six human cancer cell lines of different origins. In the absence of IMP-3, the levels of CCND1, D3 and G1 proteins fall dramatically, and the cells accumulate in the G1 phase of the cell cycle, leading to almost complete proliferation arrest. Our results show that, compared with IMP-1 and IMP-2, IMP-3 is enriched in the nucleus, where it binds the transcripts of CCND1, D3 and G1. The nuclear localization of IMP-3 depends on its protein partner HNRNPM and is indispensable for the post-transcriptional regulation of expression of the cyclins. Cytoplasmic retention of IMP-3 and HNRNPM in human cancer cells leads to significant drop in proliferation. In conclusion, a nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3 and G1 and for the proliferation of human cancer cells.

  2. The post-transcriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts.

    Science.gov (United States)

    Lucchetti-Miganeh, Céline; Burrowes, Elizabeth; Baysse, Christine; Ermel, Gwennola

    2008-01-01

    The importance of Csr post-transcriptional systems is gradually emerging; these systems control a variety of virulence-linked physiological traits in many pathogenic bacteria. This review focuses on the central role that Csr systems play in the pathogenesis of certain bacteria and in the establishment of successful infections in animal hosts. Csr systems appear to control the 'switch' between different physiological states in the infection process; for example switching pathogens from a colonization state to a persistence state. Csr systems are controlled by two-component sensor/regulator systems and by non-coding RNAs. In addition, recent findings suggest that the RNA chaperone Hfq may play an integral role in Csr-mediated bacterial adaptation to the host environment.

  3. piRNAs from Pig Testis Provide Evidence for a Conserved Role of the Piwi Pathway in Post-Transcriptional Gene Regulation in Mammals.

    Science.gov (United States)

    Gebert, Daniel; Ketting, René F; Zischler, Hans; Rosenkranz, David

    2015-01-01

    Piwi-interacting (pi-) RNAs guide germline-expressed Piwi proteins in order to suppress the activity of transposable elements (TEs). But notably, the majority of pachytene piRNAs in mammalian testes is not related to TEs. This raises the question of whether the Piwi/piRNA pathway exerts functions beyond TE silencing. Although gene-derived piRNAs were described many times, a possible gene-regulatory function was doubted due to the absence of antisense piRNAs. Here we sequenced and analyzed piRNAs expressed in the adult testis of the pig, as this taxon possesses the full set of mammalian Piwi paralogs while their spermatozoa are marked by an extreme fitness due to selective breeding. We provide an exhaustive characterization of porcine piRNAs and genomic piRNA clusters. Moreover, we reveal that both sense and antisense piRNAs derive from protein-coding genes, while exhibiting features that clearly show that they originate from the Piwi/piRNA-mediated post-transcriptional silencing pathway, commonly referred to as ping-pong cycle. We further show that the majority of identified piRNA clusters in the porcine genome spans exonic sequences of protein-coding genes or pseudogenes, which reveals a mechanism by which primary antisense piRNAs directed against mRNA can be generated. Our data provide evidence that spliced mRNAs, derived from such loci, are not only targeted by piRNAs but are also subject to ping-pong cycle processing. Finally, we demonstrate that homologous genes are targeted and processed by piRNAs in pig, mouse and human. Altogether, this strongly suggests a conserved role for the mammalian Piwi/piRNA pathway in post-transcriptional regulation of protein-coding genes, which did not receive much attention so far.

  4. Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages.

    Science.gov (United States)

    Basler, Tina; Jeckstadt, Sabine; Valentin-Weigand, Peter; Goethe, Ralph

    2006-03-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

  5. Post-transcriptional regulation of the GAP-43 gene by specific sequences in the 3' untranslated region of the mRNA.

    Science.gov (United States)

    Tsai, K C; Cansino, V V; Kohn, D T; Neve, R L; Perrone-Bizzozero, N I

    1997-03-15

    We have shown previously that GAP-43 gene expression during neuronal differentiation is controlled by selective changes in mRNA stability. This process was found to depend on highly conserved sequences in the 3' untranslated region (3' UTR) of the mRNA. To map the sequences in the GAP-43 3' UTR that mediate this post-transcriptional event, we generated specific 3' UTR deletion mutants and chimeras with the beta-globin gene and measured their half-lives in transfected PC12 cells. Our results indicate that there are two distinct instability-conferring elements localized at the 5' and 3' ends of the GAP-43 3' UTR. Of these destabilizing elements, only the one at the 3' end is required for the stabilization of the mRNA in response to treatment with the phorbol ester TPA. This 3' UTR element consists of highly conserved uridine-rich sequences and contains specific recognition sites for two neural-specific GAP-43 mRNA-binding proteins. Analysis of the levels of mRNA and protein derived from various 3' UTR deletion mutants indicated that all mutants were translated effectively and that differences in gene expression in response to TPA were attributable to changes in GAP-43 mRNA stability. In addition, the phorbol ester was found to affect the binding of specific RNA-binding proteins to the 3' UTR of the GAP-43 mRNA. Given that, like the GAP-43 mRNA, its degradation machinery and the GAP-43 mRNA-binding proteins are expressed primarily in neural cells, we propose that these factors may be involved in the post-transcriptional regulation of GAP-43 gene expression during neuronal differentiation.

  6. Interconnection of post-transcriptional regulation: The RNA-binding protein Hfq is a novel target of the Lon protease in Pseudomonas aeruginosa.

    Science.gov (United States)

    Fernández, Lucía; Breidenstein, Elena B M; Taylor, Patrick K; Bains, Manjeet; de la Fuente-Núñez, César; Fang, Yuan; Foster, Leonard J; Hancock, Robert E W

    2016-01-01

    Besides being a major opportunistic human pathogen, Pseudomonas aeruginosa can be found in a wide range of environments. This versatility is linked to complex regulation, which is achieved through the action of transcriptional regulators, and post-transcriptional regulation by intracellular proteases including Lon. Indeed, lon mutants in this species show defects in motility, biofilm formation, pathogenicity and fluoroquinolone resistance. Here, the proteomic approach stable isotope labeling by amino acids in cell culture (SILAC) was used to search for novel proteolytic targets. One of the proteins that accumulated in the lon mutant was the RNA-binding protein Hfq. Further experiments demonstrated the ability of Lon to degrade Hfq in vitro. Also, overexpression of the hfq gene in the wild-type strain led to partial inhibition of swarming, swimming and twitching motilities, indicating that Hfq accumulation could contribute to the phenotypes displayed by Lon mutants. Hfq overexpression also led to the upregulation of the small regulatory RNA PhrS. Analysis of the phenotypes of strains lacking or overexpressing this sRNA indicated that the Lon protease might be indirectly regulating the levels and activity of sRNAs via Hfq. Overall, this study revealed new links in the complex regulatory chain that controls multicellular behaviours in P. aeruginosa.

  7. Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle.

    Directory of Open Access Journals (Sweden)

    Vignesh Kumar Gunasekharan

    2016-07-01

    Full Text Available Human papillomaviruses (HPVs are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6-directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including TCHHL1, VIM, ACTN1, and POT1, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV replication

  8. MECP2 Is Post-transcriptionally Regulated during Human Neurodevelopment by Combinatorial Action of RNA-Binding Proteins and miRNAs

    Directory of Open Access Journals (Sweden)

    Deivid C. Rodrigues

    2016-10-01

    Full Text Available A progressive increase in MECP2 protein levels is a crucial and precisely regulated event during neurodevelopment, but the underlying mechanism is unclear. We report that MECP2 is regulated post-transcriptionally during in vitro differentiation of human embryonic stem cells (hESCs into cortical neurons. Using reporters to identify functional RNA sequences in the MECP2 3′ UTR and genetic manipulations to explore the role of interacting factors on endogenous MECP2, we discover combinatorial mechanisms that regulate RNA stability and translation. The RNA-binding protein PUM1 and pluripotent-specific microRNAs destabilize the long MECP2 3′ UTR in hESCs. Hence, the 3′ UTR appears to lengthen during differentiation as the long isoform becomes stable in neurons. Meanwhile, translation of MECP2 is repressed by TIA1 in hESCs until HuC predominates in neurons, resulting in a switch to translational enhancement. Ultimately, 3′ UTR-directed translational fine-tuning differentially modulates MECP2 protein in the two cell types to levels appropriate for normal neurodevelopment.

  9. Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs

    DEFF Research Database (Denmark)

    Nepal, Chirag; Coolen, Marion; Hadzhiev, Yavor;

    2015-01-01

    MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we...... used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post....... Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre...

  10. Splicing Machinery Facilitates Post-Transcriptional Regulation by FBFs and Other RNA-Binding Proteins in Caenorhabditis elegans Germline.

    Science.gov (United States)

    Novak, Preston; Wang, Xiaobo; Ellenbecker, Mary; Feilzer, Sara; Voronina, Ekaterina

    2015-08-11

    Genetic interaction screens are an important approach for understanding complex regulatory networks governing development. We used a genetic interaction screen to identify cofactors of FBF-1 and FBF-2, RNA-binding proteins that regulate germline stem cell proliferation in Caenorhabditis elegans. We found that components of splicing machinery contribute to FBF activity as splicing factor knockdowns enhance sterility of fbf-1 and fbf-2 single mutants. This sterility phenocopied multiple aspects of loss of fbf function, suggesting that splicing factors contribute to stem cell maintenance. However, previous reports indicate that splicing factors instead promote the opposite cell fate, namely, differentiation. We explain this discrepancy by proposing that splicing factors facilitate overall RNA regulation in the germline. Indeed, we find that loss of splicing factors produces synthetic phenotypes with a mutation in another RNA regulator, FOG-1, but not with a mutation in a gene unrelated to posttranscriptional regulation (dhc-1). We conclude that inefficient pre-mRNA splicing may interfere with multiple posttranscriptional regulatory events, which has to be considered when interpreting results of genetic interaction screens.

  11. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis

    Science.gov (United States)

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control caro...

  12. Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

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    Miyazawa Haruna

    2012-09-01

    Full Text Available Abstract Background Periodontal disease is suggested to increase the risk of atherothrombotic disease by inducing dyslipidemia. Recently, we demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9, which is known to play a critical role in the regulation of circulating low-density lipoprotein (LDL cholesterol levels, is elevated in periodontitis patients. However, the underlying mechanisms of elevation of PCSK9 in periodontitis patients are largely unknown. Here, we explored whether Porphyromonas gingivalis, a representative periodontopathic bacterium, -induced inflammatory response regulates serum PCSK9 and cholesterol levels using animal models. Methods We infected C57BL/6 mice intraperitoneally with Porphyromonas gingivalis, a representative strain of periodontopathic bacteria, and evaluated serum PCSK9 levels and the serum lipid profile. PCSK9 and LDL receptor (LDLR gene and protein expression, as well as liver X receptors (Lxrs, inducible degrader of the LDLR (Idol, and sterol regulatory element binding transcription factor (Srebf2 gene expression, were examined in the liver. Results P. gingivalis infection induced a significant elevation of serum PCSK9 levels and a concomitant elevation of total and LDL cholesterol compared with sham-infected mice. The LDL cholesterol levels were significantly correlated with PCSK9 levels. Expression of the Pcsk9, Ldlr, and Srebf2 genes was upregulated in the livers of the P. gingivalis-infected mice compared with the sham-infected mice. Although Pcsk9 gene expression is known to be positively regulated by sterol regulatory element binding protein (SREBP2 (human homologue of Srebf2, whereas Srebf2 is negatively regulated by cholesterol, the elevated expression of Srebf2 found in the infected mice is thought to be mediated by P. gingivalis infection. Conclusions P. gingivalis infection upregulates PCSK9 production via upregulation of Srebf2, independent of cholesterol levels. Further studies

  13. Post-transcriptional regulation of coumarin 7-hydroxylase (P450coh) induction by xenobiotics in mouse liver: mRNA stabilization by pyrazole

    Energy Technology Data Exchange (ETDEWEB)

    Aida, K.; Negishi, M. (NIEHS/NIH, Research Triangle Park, NC (United States))

    1991-03-15

    The induction mechanism by pyrazole or phenobarbital of coumarin 7-hydroxylase was investigated in DBA/2J male mice. The P450coh mRNA in the pyrazole-induced mice was increased gradually to a 20-fold higher level within 48 hr, yet transcription of the P450coh gene was not affected. The half-life of P450coh mRNA, on the other hand, was at least 4-fold longer in the pyrazole-induced DBA2J than in control DBA/2J male mice. The stabilization of P450coh mRNA, therefore, is the primary mechanism for the induction by pyrazole of coumarin 7-hydroxylase. Phenobarbital, on the other hand, regulates the induction translationally or post-translationally. This drug affected neither the P450coh mRNA nor the P450coh gene's transcription levels in the DBA/2J male mice, although Western blots showed a 2- to 3-fold increase of the P450coh protein in the liver microsomes of the drug-treated mice. The results indicate, therefore, that both phenobarbital and pyrazole regulate the P450coh induction post-transcriptional efficiency of P450coh mRNA or alters the degradation rate of P450coh protein, while the latter stabilizes P450coh mRNA.

  14. Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells.

    Science.gov (United States)

    Miles, Wayne O; Korenjak, Michael; Griffiths, Lyra M; Dyer, Michael A; Provero, Paolo; Dyson, Nicholas J

    2014-10-01

    Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE).

  15. Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa

    Science.gov (United States)

    Reales-Calderón, Jose Antonio; Corona, Fernando; Monteoliva, Lucía; Gil, Concha; Martínez, Jose Luis

    2015-01-01

    Crc is a post-transcriptional regulator in Pseudomonas aeruginosa that modulates its metabolism, but also its susceptibility to antibiotics and virulence. Most of P. aeruginosa virulence factors are secreted or engulfed in vesicles. A Crc deficient mutant was created and the extracellular vesicles associated exoproteome and the vesicle-free secretome was quantified using iTRAQ. Fifty vesicles-associated proteins were more abundant and 14 less abundant in the Crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Different virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the Crc-defective mutant. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain, in agreement with the low secretion of proteins related to virulence. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals. PMID:26306318

  16. Integrated analysis of seed proteome and mRNA oxidation reveals distinct post-transcriptional features regulating dormancy in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Gao, Feng; Rampitsch, Christof; Chitnis, Vijaya R; Humphreys, Gavin D; Jordan, Mark C; Ayele, Belay T

    2013-10-01

    Wheat seeds can be released from a dormant state by after-ripening; however, the underlying molecular mechanisms are still mostly unknown. We previously identified transcriptional programmes involved in the regulation of after-ripening-mediated seed dormancy decay in wheat (Triticum aestivum L.). Here, we show that seed dormancy maintenance and its release by dry after-ripening in wheat is associated with oxidative modification of distinct seed-stored mRNAs that mainly correspond to oxidative phosphorylation, ribosome biogenesis, nutrient reservoir and α-amylase inhibitor activities, suggesting the significance of post-transcriptional repression of these biological processes in regulating seed dormancy. We further show that after-ripening induced seed dormancy release in wheat is mediated by differential expression of specific proteins in both dry and hydrated states, including those involved in proteolysis, cellular signalling, translation and energy metabolism. Among the genes corresponding to these proteins, the expression of those encoding α-amylase/trypsin inhibitor and starch synthase appears to be regulated by mRNA oxidation. Co-expression analysis of the probesets differentially expressed and oxidized during dry after-ripening along with those corresponding to proteins differentially regulated between dormant and after-ripened seeds produced three co-expressed gene clusters containing more candidate genes potentially involved in the regulation of seed dormancy in wheat. Two of the three clusters are enriched with elements that are either abscisic acid (ABA) responsive or recognized by ABA-regulated transcription factors, indicating the association between wheat seed dormancy and ABA sensitivity.

  17. Regulation of the CDP-choline pathway by sterol regulatory element binding proteins involves transcriptional and post-transcriptional mechanisms.

    Science.gov (United States)

    Ridgway, Neale D; Lagace, Thomas A

    2003-06-15

    The synthesis of phosphatidylcholine (PtdCho) by the CDP-choline pathway is under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). Sterol regulatory element binding proteins (SREBPs) have been proposed to regulate CCT at the transcriptional level, or via the synthesis of lipid activators or substrates of the CDP-choline pathway. To assess the contributions of these two mechanisms, we examined CCTalpha expression and PtdCho synthesis by the CDP-choline pathway in cholesterol and fatty acid auxotrophic CHO M19 cells inducibly expressing constitutively active nuclear forms of SREBP1a or SREBP2. Induction of either SREBP resulted in increased expression of mRNAs for sterol-regulated genes, elevated fatty acid and cholesterol synthesis (>10-50-fold) and increased PtdCho synthesis (2-fold). CCTalpha mRNA was increased 2-fold by enforced expression of SREBP1a or SREBP2. The resultant increase in CCTalpha protein and activity (2-fold) was restricted primarily to the soluble fraction of cells, and increased CCTalpha activity in vivo was not detected. Inhibition of the synthesis of fatty acids or their CoA esters by cerulenin or triacsin C respectively following SREBP induction effectively blocked the accompanying elevation in PtdCho synthesis. Thus PtdCho synthesis was driven by increased synthesis of fatty acids or a product thereof. These data show that transcriptional activation of CCTalpha is modest relative to that of other SREBP-regulated genes, and that stimulation of PtdCho synthesis by SREBPs in CHO cells is due primarily to increased fatty acid synthesis.

  18. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1.

    Directory of Open Access Journals (Sweden)

    Goutam Mandal

    2015-02-01

    Full Text Available Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V acts as a pro-drug, which is converted to the more active trivalent form (Sb(III. However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL are more sensitive to Sb(III than the species responsible for visceral leishmaniasis (VL. Leishmania aquaglyceroporin (AQP1 facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3'-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.

  19. Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

    Science.gov (United States)

    Mandal, Goutam; Mandal, Srotoswati; Sharma, Mansi; Charret, Karen Santos; Papadopoulou, Barbara; Bhattacharjee, Hiranmoy; Mukhopadhyay, Rita

    2015-01-01

    Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species. PMID:25714343

  20. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  1. The L1TD1 protein interactome reveals the importance of post-transcriptional regulation in human pluripotency.

    Science.gov (United States)

    Emani, Maheswara Reddy; Närvä, Elisa; Stubb, Aki; Chakroborty, Deepankar; Viitala, Miro; Rokka, Anne; Rahkonen, Nelly; Moulder, Robert; Denessiouk, Konstantin; Trokovic, Ras; Lund, Riikka; Elo, Laura L; Lahesmaa, Riitta

    2015-03-10

    The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  2. Berberine Induces Apoptosis in p53-Null Leukemia Cells by Down-Regulating XIAP at the Post-Transcriptional Level

    Directory of Open Access Journals (Sweden)

    Jian Liu

    2013-11-01

    Full Text Available Background: Berberine exerts anticancer activities both in vitro and in vivo through different mechanisms. However, the underlying molecular mechanisms of berberine induced p53-independent apoptosis remain unclear. Methods: The p53-null leukemia cell line EU-4 cells were exposed to berberine. Then the cell viability and apoptosis were determined. Western blot and PCR were employed to detect the expression of apoptosis related protein, XIAP and MDM2. Small interfering RNA (siRNA was applied to knock down endogenous expression of MDM2 and XIAP. Results: Berberine induced p53-independent, XIAP-mediated apoptotic cell death in p53-null leukemia cells. Treatment with berberine resulted in suppression of XIAP protein in a dose- and time- dependent manner. Berberine induced down-regulation of XIAP protein involving inhibition of MDM2 expression and a proteasome-dependent pathway. Moreover, inhibition of XIAP by berberine or siRNA increased the sensitivity of leukemia cells to doxorubicin-induced apoptosis. Conclusion: Our findings characterize the molecular mechanisms of berberine-induced caspase activation and subsequent apoptosis, and berberine may be a novel candidate inducer of apoptosis in leukemia cells, which normally lack p53 expression.

  3. Heat Stress Regulates the Expression of Genes at Transcriptional and Post-Transcriptional Levels, Revealed by RNA-seq in Brachypodium distachyon

    Science.gov (United States)

    Chen, Shoukun; Li, Haifeng

    2017-01-01

    Heat stress greatly affects plant growth/development and influences the output of crops. With the increased occurrence of extreme high temperature, the negative influence on cereal products from heat stress becomes severer and severer. It is urgent to reveal the molecular mechanism in response to heat stress in plants. In this research, we used RNA-seq technology to identify differentially expressed genes (DEGs) in leaves of seedlings, leaves and inflorescences at heading stage of Brachypodium distachyon, one model plant of grasses. Results showed many genes in responding to heat stress. Of them, the expression level of 656 DEGs were altered in three groups of samples treated with high temperature. Gene ontology (GO) analysis showed that the highly enriched DEGs were responsible for heat stress and protein folding. According to KEGG pathway analysis, the DEGs were related mainly to photosynthesis-antenna proteins, the endoplasmic reticulum, and the spliceosome. Additionally, the expression level of 454 transcription factors belonging to 49 gene families was altered, as well as 1,973 splicing events occurred after treatment with high temperature. This research lays a foundation for characterizing the molecular mechanism of heat stress response and identifying key genes for those responses in plants. These findings also clearly show that heat stress regulates the expression of genes not only at transcriptional level, but also at post-transcriptional level. PMID:28119730

  4. Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs.

    Science.gov (United States)

    Nepal, Chirag; Coolen, Marion; Hadzhiev, Yavor; Cussigh, Delphine; Mydel, Piotr; Steen, Vidar M; Carninci, Piero; Andersen, Jesper B; Bally-Cuif, Laure; Müller, Ferenc; Lenhard, Boris

    2016-04-20

    MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9-5 was validated by 5' RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3'-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay.

  5. Post-transcriptional gene silencing across kingdoms.

    Science.gov (United States)

    Cogoni, C; Macino, G

    2000-12-01

    Post-transcriptional gene silencing (PTGS) as a consequence of the introduction of either transgenes or double-stranded RNA molecules has been found to occur in a number of species. In the past year, studies in different systems have greatly enhanced our understanding of the molecular mechanisms of these phenomena. The ubiquitous presence of PTGS in both the plant and animal kingdoms and the finding of common genetic mechanisms suggest that PTGS is a universal gene-regulation system fundamental in biological processes such as protection against viruses and transposons.

  6. The post-transcriptional regulator rsmA/csrA activates T3SS by stabilizing the 5' UTR of hrpG, the master regulator of hrp/hrc genes, in Xanthomonas.

    Directory of Open Access Journals (Sweden)

    Maxuel O Andrade

    2014-02-01

    Full Text Available The RsmA/CsrA family of the post-transcriptional regulators of bacteria is involved in the regulation of many cellular processes, including pathogenesis. In this study, we demonstrated that rsmA not only is required for the full virulence of the phytopathogenic bacterium Xanthomonas citri subsp. citri (XCC but also contributes to triggering the hypersensitive response (HR in non-host plants. Deletion of rsmA resulted in significantly reduced virulence in the host plant sweet orange and a delayed and weakened HR in the non-host plant Nicotiana benthamiana. Microarray, quantitative reverse-transcription PCR, western-blotting, and GUS assays indicated that RsmA regulates the expression of the type 3 secretion system (T3SS at both transcriptional and post-transcriptional levels. The regulation of T3SS by RsmA is a universal phenomenon in T3SS-containing bacteria, but the specific mechanism seems to depend on the interaction between a particular bacterium and its hosts. For Xanthomonads, the mechanism by which RsmA activates T3SS remains unknown. Here, we show that RsmA activates the expression of T3SS-encoding hrp/hrc genes by directly binding to the 5' untranslated region (UTR of hrpG, the master regulator of the hrp/hrc genes in XCC. RsmA stabilizes hrpG mRNA, leading to increased accumulation of HrpG proteins and subsequently, the activation of hrp/hrc genes. The activation of the hrp/hrc genes by RsmA via HrpG was further supported by the observation that ectopic overexpression of hrpG in an rsmA mutant restored its ability to cause disease in host plants and trigger HR in non-host plants. RsmA also stabilizes the transcripts of another T3SS-associated hrpD operon by directly binding to the 5' UTR region. Taken together, these data revealed that RsmA primarily activates T3SS by acting as a positive regulator of hrpG and that this regulation is critical to the pathogenicity of XCC.

  7. Post-transcriptional and post-translational regulations of drought and heat response in plants: a spider’s web of mechanisms

    Directory of Open Access Journals (Sweden)

    Davide eGuerra

    2015-02-01

    Full Text Available Drought and heat tolerance are complex quantitative traits. Moreover, the adaptive significance of some stress-related traits is more related to plant survival than to agronomic performance. A web of regulatory mechanisms fine-tunes the expression of stress-related traits and integrates both environmental and developmental signals. Both post-transcriptional and post-translational modifications contribute substantially to this network with a pivotal regulatory function of the transcriptional changes related to cellular and plant stress response. Alternative splicing and RNA-mediated silencing control the amount of specific transcripts, while ubiquitin and SUMO modify activity, sub-cellular localization and half-life of proteins. Interactions across these modification mechanisms ensure temporally and spatially appropriate patterns of downstream-gene expression. For key molecular components of these regulatory mechanisms, natural genetic diversity exists among genotypes with different behavior in terms of stress tolerance, with effects upon the expression of adaptive morphological and/or physiological target traits.

  8. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of h......-life of osteoblastic PDGFR-alpha mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-alpha is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism.......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation...... of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  9. Sodium-dependent phosphate uptake in the jejunum is post-transcriptionally regulated in pigs fed a low-phosphorus diet and is independent of dietary calcium concentration.

    Science.gov (United States)

    Saddoris, Kari L; Fleet, James C; Radcliffe, John S

    2010-04-01

    In rodents, severe dietary P restriction increases active phosphate absorption by the intestine. However, it remains unknown if moderate dietary P restriction has a similar effect. Weanling pigs (n = 32; body weight 7.4 +/- 0.55 kg) were used in a 2 x 2 factorial design and fed dietary available P (aP) concentrations of 0.23 or 0.40% and Ca concentrations of 0.58 or 1.00% for 14 d. Diets were formulated on an aP basis instead of a total P basis, because pigs are unable to absorb phytate-P present in corn and soybean meal. Jejunal segments were mounted in modified Ussing chambers for determination of Na(+)-dependent nutrient transport. Intestinal mucosal scrapings were taken for RNA isolation and brush border membrane (BBM) vesicle isolation. Na(+)-dependent phosphate uptake and gene expression of Na-phosphate cotransporter IIb (NaPi-IIb), SGLT-1 (sodium/glucose cotransporter-1), and calbindin D(9k) and protein expression of NaPi-IIb were evaluated. Na(+)-dependent phosphate transport increased (P dietary aP concentration was decreased. However, increased Na(+)-dependent phosphate uptake was not accompanied by increased NaPi-IIb mRNA expression. Expression of NaPi-IIb protein in the BBM increased (P pigs fed low-P diets compared with pigs fed adequate-P diets. No dietary Ca effects or aP x Ca interactions were detected for Na-dependent P uptake, mRNA or protein expression of NaPi-IIb, or mRNA expression of calbindin D(9k). These data suggest that restricting dietary aP concentration by only 43% stimulates Na(+)-dependent phosphate uptake and expression of the NaPi-IIb protein in the BBM of the small intestine and through a post-transcriptional mechanism.

  10. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells.

    Science.gov (United States)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen; Stark, G Björn

    2010-01-01

    Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR-alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells but not in immortalized osteoblastic cell lines. Functional inhibition of gap junctional communication between HUVECs and hOBs by 18alpha-glycyrrhetinic acid had no effect on HUVEC-mediated PDGFR-alpha downregulation, whereas inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the HUVEC-mediated reduction in osteoblastic PDGFR-alpha expression. To delineate the molecular mechanism underlying the PDGFR-alpha downregulation, we examined the effect of HUVEC co-cultivation on osteoblastic PDGFR-alpha promoter activity as well as mRNA stability. Co-cultivation of HUVECs with hOBs significantly shortened the half-life of osteoblastic PDGFR-alpha mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-alpha is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism.

  11. Loss of post-transcriptional regulation of DNMT3b by microRNAs: a possible molecular mechanism for the hypermethylation defect observed in a subset of breast cancer cell lines.

    Science.gov (United States)

    Sandhu, Rupninder; Rivenbark, Ashley G; Coleman, William B

    2012-08-01

    A hypermethylation defect associated with DNMT hyperactivity and DNMT3b overexpression characterizes a subset of breast cancers and breast cancer cell lines. We analyzed breast cancer cell lines for differential expression of regulatory miRs to determine if loss of miR-mediated post-transcriptional regulation of DNMT3b represents the molecular mechanism that governs the overexpression of DNMT3b that drives the hypermethylation defect in breast cancer. MicroRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a, miR-148b) or are predicted (miR-26a, miR-26b, miR-203, miR-222) to regulate DNMT3b were examined among 10 hypermethylator and 6 non-hypermethylator breast cancer cell lines. Hypermethylator cell lines express diminished levels of miR-29c, miR-148a, miR-148b, miR-26a, miR-26b, and miR-203 compared to non-hypermethylator cell lines. miR expression patterns correlate inversely with methylation-sensitive gene expression (r=-0.66, p=0.0056) and directly with the methylation status of these genes (r=0.72, p=0.002). To determine the mechanistic role of specific miRs in the dysregulation of DNMT3b among breast cancer cell lines, miR levels were modulated by transfection of pre-miR precursors for miR-148b, miR-26b, and miR-29c into hypermethylator cell lines (Hs578T, HCC1937, SUM185) and transfection of antagomirs directed against miR-148b, miR-26b, and miR-29c into non-hypermethylator cell lines (BT20, MDA-MB-415, MDA-MB-468). Antagomir-mediated knock-down of miR-148b, miR-29c, and miR-26b significantly increased DNMT3b mRNA in non-hypermethylator cell lines, and re-expression of miR-148b, miR-29c, and miR-26b following transfection of pre-miR precursors significantly reduced DNMT3b mRNA in hypermethylator cell lines. These findings strongly suggest that: i) post-transcriptional regulation of DNMT3b is combinatorial, ii) diminished expression of regulatory miRs contributes to DNMT3b overexpression, iii) re-expression of regulatory miRs reduces DNMT3b m

  12. The Role of the 3' Untranslated Region in the Post-Transcriptional Regulation of KLF6 Gene Expression in Hepatocellular Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Diab, Thoria; Hanoun, Naima [INSERM UMR 1037, Toulouse 31432 (France); Paul Sabatier University, Toulouse 31000 (France); Bureau, Christophe; Christol, Camille [INSERM UMR 1037, Toulouse 31432 (France); Paul Sabatier University, Toulouse 31000 (France); Department of Hepatology, Toulouse University Hospital Centre, Toulouse 31409 (France); Buscail, Louis [INSERM UMR 1037, Toulouse 31432 (France); Paul Sabatier University, Toulouse 31000 (France); Department of Gastroenterology, Toulouse University Hospital Centre, Toulouse 31409 (France); Cordelier, Pierre, E-mail: pierre.cordelier@inserm.fr; Torrisani, Jérôme [INSERM UMR 1037, Toulouse 31432 (France); Paul Sabatier University, Toulouse 31000 (France)

    2013-12-19

    KLF6 is ubiquitously expressed in human tissues and regulates many pathways such as differentiation, development, cellular proliferation, growth-related signal transduction, and apoptosis. We previously demonstrated that KLF6 expression is altered during liver carcinogenesis. More importantly, KLF6 invalidation results in cell cycle progression inhibition and apoptosis of liver cancer cells. On the other hand, enforced expression of KLF6 variant 2 (SV2) induces cancer cell death by apoptosis. Thus, we and others demonstrated that KLF6 and its splicing variants play a critical role in liver cancer. However, little is known on the mechanisms governing KLF6 expression in HCC. In the present work, we asked whether the 3' untranslated region (3'UTR) of the KLF6 mRNA may be responsible for regulation of KLF6 expression in HCC. We found that KLF6 mRNA stability was altered in liver-derived cell lines as compared to cervical cancer-derived cell lines and human embryonic fibroblasts. Interestingly, KLF6 mRNA was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes. We next cloned the KLF6 mRNA 3'UTR into luciferase-expressing vectors and found that gene expression and activity were strongly impaired in all liver-derived cell lines tested. In addition, we found that most the KLF6 3'UTR destabilisation activity resides between nt 1,835 and nt 2,615 of the KLF6 gene. Taken together, we provide the first steps towards better understanding of the regulation of KLF6 expression in HCC. Further work is needed to identify the factors that bind to KLF6 3'UTR to regulate its expression in liver cancer-derived cell lines.

  13. Transcriptional and post-transcriptional profile of human chromosome 21.

    Science.gov (United States)

    Nikolaev, Sergey I; Deutsch, Samuel; Genolet, Raphael; Borel, Christelle; Parand, Leila; Ucla, Catherine; Schütz, Frederic; Duriaux Sail, Genevieve; Dupré, Yann; Jaquier-Gubler, Pascale; Araud, Tanguy; Conne, Beatrice; Descombes, Patrick; Vassalli, Jean-Dominique; Curran, Joseph; Antonarakis, Stylianos E

    2009-08-01

    Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that approximately 50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that approximately 40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

  14. Multi-Faceted Post-Transcriptional Functions of HIV-1 Rev

    Directory of Open Access Journals (Sweden)

    Kuan-Teh Jeang

    2012-07-01

    Full Text Available Post-transcriptional regulation of HIV-1 gene expression is largely governed by the activities of the viral Rev protein. In this minireview, the multiple post-transcriptional activities of Rev in the export of partially spliced and unspliced HIV-1 RNAs from the nucleus to the cytoplasm, in the translation of HIV-1 transcripts, and in the packaging of viral genomic RNAs are reviewed in brief.

  15. miR-200b mediates post-transcriptional repression of ZFHX1B

    DEFF Research Database (Denmark)

    Christoffersen, Nanna Rønbjerg; Silahtaroglu, Asli; Ørom, Ulf Lupo Andersson

    2007-01-01

    MicroRNAs have important functions during animal development and homeostasis through post-transcriptional regulation of their cognate mRNA targets. ZFHX1B is a transcriptional repressor involved in the TGFbeta signaling pathway and in processes of epithelial to mesenchymal transition via regulati...

  16. AURA 2: Empowering discovery of post-transcriptional networks.

    Science.gov (United States)

    Dassi, Erik; Re, Angela; Leo, Sara; Tebaldi, Toma; Pasini, Luigi; Peroni, Daniele; Quattrone, Alessandro

    2014-01-01

    Post-transcriptional regulation (PTR) of gene expression is now recognized as a major determinant of cell phenotypes. The recent availability of methods to map protein-RNA interactions in entire transcriptomes such as RIP, CLIP and their variants, together with global polysomal and ribosome profiling techniques, are driving the exponential accumulation of vast amounts of data on mRNA contacts in cells, and of corresponding predictions of PTR events. However, this exceptional quantity of information cannot be exploited at its best to reconstruct potential PTR networks, as it still lies scattered throughout several databases and in isolated reports of single interactions. To address this issue, we developed the second and vastly enhanced version of the Atlas of UTR Regulatory Activity (AURA 2), a meta-database centered on mapping interaction of trans-factors with human and mouse UTRs. AURA 2 includes experimentally demonstrated binding sites for RBPs, ncRNAs, thousands of cis-elements, variations, RNA epigenetics data and more. Its user-friendly interface offers various data-mining features including co-regulation search, network generation and regulatory enrichment testing. Gene expression profiles for many tissues and cell lines can be also combined with these analyses to display only the interactions possible in the system under study. AURA 2 aims at becoming a valuable toolbox for PTR studies and at tracing the road for how PTR network-building tools should be designed. AURA 2 is available at http://aura.science.unitn.it.

  17. Honing the message: post-transcriptional and post-translational control in attaching and effacing pathogens.

    Science.gov (United States)

    Bhatt, Shantanu; Romeo, Tony; Kalman, Daniel

    2011-05-01

    Bacteria evolve their capacity to cause disease by acquiring virulence genes that are usually clustered in discrete genetic modules termed pathogenicity islands (PAI). Stable integration of PAIs into pre-existing transcriptional networks coordinates expression from PAIs with ancestral genes in response to diverse environmental cues. Such transcriptional controls are evident in the regulation of the locus of enterocyte effacement (LEE), a PAI of enteropathogenic and enterohemorrhagic Escherichia coli. However, recent reports indicate that global post-transcriptional and post-translational regulators, including CsrA, Hfq and ClpXP, fine-tune the transcriptional output from the LEE. In this opinion article, we highlight recent advances in the understanding of post-transcriptional and post-translational regulation in attaching and effacing pathogens.

  18. Complementary Post Transcriptional Regulatory Information is Detected by PUNCH-P and Ribosome Profiling

    Science.gov (United States)

    Zur, Hadas; Aviner, Ranen; Tuller, Tamir

    2016-01-01

    Two novel approaches were recently suggested for genome-wide identification of protein aspects synthesized at a given time. Ribo-Seq is based on sequencing all the ribosome protected mRNA fragments in a cell, while PUNCH-P is based on mass-spectrometric analysis of only newly synthesized proteins. Here we describe the first Ribo-Seq/PUNCH-P comparison via the analysis of mammalian cells during the cell-cycle for detecting relevant differentially expressed genes between G1 and M phase. Our analyses suggest that the two approaches significantly overlap with each other. However, we demonstrate that there are biologically meaningful proteins/genes that can be detected to be post-transcriptionally regulated during the mammalian cell cycle only by each of the approaches, or their consolidation. Such gene sets are enriched with proteins known to be related to intra-cellular signalling pathways such as central cell cycle processes, central gene expression regulation processes, processes related to chromosome segregation, DNA damage, and replication, that are post-transcriptionally regulated during the mammalian cell cycle. Moreover, we show that combining the approaches better predicts steady state changes in protein abundance. The results reported here support the conjecture that for gaining a full post-transcriptional regulation picture one should integrate the two approaches. PMID:26898226

  19. High SINE RNA Expression Correlates with Post-Transcriptional Downregulation of BRCA1

    Directory of Open Access Journals (Sweden)

    Giovanni Bosco

    2013-04-01

    Full Text Available Short Interspersed Nuclear Elements (SINEs are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is unknown. Here, we show that downregulation of the mRNA of the tumor suppressor gene BRCA1 is associated with increased transcription of SINEs and production of sense and antisense SINE small RNAs. We find that BRCA1 mRNA is post-transcriptionally down-regulated in a Dicer and Drosha dependent manner and that expression of a SINE inverted repeat with sequence identity to a BRCA1 intron is sufficient for downregulation of BRCA1 mRNA. These observations suggest that transcriptional activation of SINEs could contribute to a novel mechanism of RNA mediated post-transcriptional silencing of human genes.

  20. PSD-95 is post-transcriptionally repressed during early neural development by PTBP1 and PTBP2

    DEFF Research Database (Denmark)

    Zheng, Sika; Gray, Erin E; Chawla, Geetanjali

    2012-01-01

    Postsynaptic density protein 95 (PSD-95) is essential for synaptic maturation and plasticity. Although its synaptic regulation has been widely studied, the control of PSD-95 cellular expression is not understood. We found that Psd-95 was controlled post-transcriptionally during neural development...

  1. MicroRNA-98 and microRNA-214 post-transcriptionally regulate enhancer of zeste homolog 2 and inhibit migration and invasion in human esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Huang Sheng-Dong

    2012-08-01

    Full Text Available Abstract Background The enhancer of zeste homolog 2 (EZH2 was found to be overexpressed and associated with tumor metastasis in esophageal squamous cell carcinoma (ESCC. On the other hand, it was reported that miR-26a, miR-98, miR-101, miR-124, miR-138 and miR-214 could inhibit the expression of EZH2 in some tumors. However, the role of miRNAs in the regulation of EZH2 expression in human ESCC has not been documented. The aim of this study was to determine the role of these miRNAs in the regulation of tumor metastasis via EZH2 overexpression in human ESCC. Methods and results The expression of these miRNAs and EZH2 mRNA were examined by qPCR and the expression of EZH2 protein was detected by western blot. The role of these miRNAs in migration and invasion was studied in ESCC cell line (Eca109 transfected with miRNA mimics or cotransfected with miRNA mimics and pcDNA-EZH2 plasmid (without the 3’-UTR of EZH2. Through clinical investigation, we found that miR-98 and miR-214 expression was significantly lower in ESCC tissues than in matched normal tissues, and the expression level of miR-98 and miR-214 was inversely correlated to EZH2 protein expression and the clinical features such as pathological grade, tumor stage and lymph node metastasis in ESCC. In Eca109 cells, overexpression of miR-98 and miR-214 significantly inhibited the migration and invasion of ESCC cells, which was reversed by transfection of EZH2. Conclusions These findings suggest that decreased expression of miR-98 and miR-214 might promote metastasis of human ESCC by inducing accumulation of EZH2 protein.

  2. The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

    Science.gov (United States)

    Morin, Manon; Ropers, Delphine; Letisse, Fabien; Laguerre, Sandrine; Portais, Jean-Charles; Cocaign-Bousquet, Muriel; Enjalbert, Brice

    2016-05-01

    Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated. Here, a multiscale analysis was performed in a wild-type strain and its isogenic mutant attenuated for CsrA (including growth parameters, gene expression levels, metabolite pools, abundance of enzymes and fluxes). Data integration and regulation analysis showed a coordinated control of the expression of glycolytic enzymes. This also revealed the imbalance of metabolite pools in the csrA mutant upper glycolysis, before the phosphofructokinase PfkA step. This imbalance is associated with a glucose-phosphate stress. Restoring PfkA activity in the csrA mutant strain suppressed this stress and increased the mutant growth rate on glucose. Thus, the carbon storage regulator system is essential for the effective functioning of the upper glycolysis mainly through its control of PfkA. This work demonstrates the pivotal role of post-transcriptional regulation to shape the carbon metabolism.

  3. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Smialowska, Agata, E-mail: smialowskaa@gmail.com [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Djupedal, Ingela; Wang, Jingwen [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Kylsten, Per [School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Swoboda, Peter [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Ekwall, Karl, E-mail: Karl.Ekwall@ki.se [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden)

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  4. Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs.

    Science.gov (United States)

    Louloupi, Annita; Ntini, Evgenia; Liz, Julia; Ørom, Ulf Andersson

    2017-03-01

    miRNAs are small regulatory RNAs involved in the regulation of translation of target transcripts. miRNA biogenesis is a multi-step process starting with the cleavage of the primary miRNA transcript in the nucleus by the Microprocessor complex. Endogenous processing of pri-miRNAs is challenging to study and the in vivo kinetics of this process is not known. Here, we present a method for determining the processing kinetics of pri-miRNAs within intact cells over time using a pulse-chase approach to label transcribed RNA during 15 minutes and follow the processing within a 1-hour window after labeling with bromouridine. We show, that pri-miRNAs exhibit different processing kinetics ranging from fast over intermediate to slow processing and provide evidence that pri-miRNA processing can occur both co-transcriptionally and post-transcriptionally.

  5. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements

    Directory of Open Access Journals (Sweden)

    Sara J.C. Gosline

    2016-01-01

    Full Text Available MicroRNAs (miRNAs regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq and CLIP (crosslinking followed by immunoprecipitation sequencing (CLIP-seq, we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.

  6. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements.

    Science.gov (United States)

    Gosline, Sara J C; Gurtan, Allan M; JnBaptiste, Courtney K; Bosson, Andrew; Milani, Pamela; Dalin, Simona; Matthews, Bryan J; Yap, Yoon S; Sharp, Phillip A; Fraenkel, Ernest

    2016-01-12

    MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.

  7. The identification of novel PMADS3 interacting proteins indicates a role in post-transcriptional control.

    Science.gov (United States)

    Li, Xin; Ning, Guogui; Han, Xueping; Liu, Caixian; Bao, Manzhu

    2015-06-10

    PMADS3, a known MADS-box transcriptional factor and a C-class gene for floral development, plays dual roles in controlling the identity of inner floral organs and the termination of flower meristems in petunia. In this study, it was confirmed by bimolecular fluorescence complementation (BiFC) assays that the PMADS3 protein can interact individually with E-class proteins FBP2, FBP5, FBP9 and PMADS12. A yeast two-hybrid cDNA library was screened using the entire PMADS3 as bait, and this identified further potential interaction candidates. Two novel genes, PheIF3f and PhAGO10, were isolated, and suggested to regulate mRNA and translational processes according to the analysis of protein functional domains and subcellular localization predictions. Notably, the PhAGO10 protein belongs to the Argonaute family, members of which are major players in small-RNA-guided gene silencing processes via mRNA cleavage or translational inhibition. The results of yeast two-hybrid and BiFC assays indicated that PheIF3f and PhAGO10 could interact with PMADS3. Our findings indicate that the C-class gene PMADS3 potentially participates in post-transcriptional control, as well as transcriptional regulation.

  8. Post-Transcriptional Control of Gene Expression in Mouse Early Embryo Development: A View from the Tip of the Iceberg

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    Claudio Sette

    2011-04-01

    Full Text Available Fertilization is a very complex biological process that requires the perfect cooperation between two highly specialized cells: the male and female gametes. The oocyte provides the physical space where this process takes place, most of the energetic need, and half of the genetic contribution. The spermatozoon mostly contributes the other half of the chromosomes and it is specialized to reach and to penetrate the oocyte. Notably, the mouse oocyte and early embryo are transcriptionally inactive. Hence, they fully depend on the maternal mRNAs and proteins stored during oocyte maturation to drive the onset of development. The new embryo develops autonomously around the four-cell stage, when maternal supplies are exhausted and the zygotic genome is activated in mice. This oocyte-to-embryo transition needs an efficient and tightly regulated translation of the maternally-inherited mRNAs, which likely contributes to embryonic genome activation. Full understanding of post-transcriptional regulation of gene expression in early embryos is crucial to understand the reprogramming of the embryonic genome, it might help driving reprogramming of stem cells in vitro and will likely improve in vitro culturing of mammalian embryos for assisted reproduction. Nevertheless, the knowledge of the mechanism(s underlying this fundamental step in embryogenesis is still scarce, especially if compared to other model organisms. We will review here the current knowledge on the post-transcriptional control of gene expression in mouse early embryos and discuss some of the unanswered questions concerning this fascinating field of biology.

  9. Elongation factor P mediates a novel post-transcriptional regulatory pathway critical for bacterial virulence

    DEFF Research Database (Denmark)

    Zou, S Betty; Roy, Hervé; Ibba, Michael;

    2012-01-01

    Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability of the path......Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability...... of the pathogen to respond to external cues are typically attenuating. Here we discuss our recent discovery of a novel post-transcriptional regulatory pathway critical for Salmonella virulence and stress resistance. The enzymes PoxA and YjeK coordinately attach a unique beta-amino acid onto a highly conserved...... our laboratory and others now suggests that EF-P, previously thought to be essential, instead plays an ancillary role in translation by regulating the synthesis of a relatively limited subset of proteins. Other observations suggest that the eukaryotic homolog of EF-P, eIF5A, may illicit similar...

  10. Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms.

    Science.gov (United States)

    Verrey, F; Pearce, D; Pfeiffer, R; Spindler, B; Mastroberardino, L; Summa, V; Zecevic, M

    2000-04-01

    The aldosterone-induced increase in sodium reabsorption across tight epithelia can be divided schematically into two functional phases: an early regulatory phase starting after a lag period of 20 to 60 minutes, during which the pre-existing transport machinery is activated, and a late phase (>2.5 h), which can be viewed as an anabolic action leading to a further amplification/differentiation of the Na+ transport machinery. At the transcriptional level, both early and late responses are initiated during the lag period, but the functional impact of newly synthesized regulatory proteins is faster than that of the structural ones. K-Ras2 and SGK were identified as the first early aldosterone-induced regulatory proteins in A6 epithelia. Their mRNAs also were shown to be regulated in vivo by aldosterone, and their expression (constitutively active K-Ras2 and wild-type SGK) was shown to increase the function of ENaC coexpressed in Xenopus oocytes. Recently, aldosterone was also shown to act on transcription factors in A6 epithelia: It down-regulates the mRNAs of the proliferation-promoting c-Myc, c-Jun, and c-Fos by a post-transcriptional mechanism, whereas it up-regulates that of Fra-2 (c-Fos antagonist) at the transcriptional level. Together, these new data illustrate the complexity of the regulatory network controlled by aldosterone and support the view that its early action is mediated by the induction of key regulatory proteins such as K-Ras2 and SGK. These early induced proteins are sites of convergence for different regulatory inputs, and thus, their aldosterone-regulated expression level tunes the impact of other regulatory cascades on sodium transport. This suggests mechanisms for the escape from aldosterone action.

  11. Accounting for experimental noise reveals that mRNA levels, amplified by post-transcriptional processes, largely determine steady-state protein levels in yeast.

    Directory of Open Access Journals (Sweden)

    Gábor Csárdi

    2015-05-01

    Full Text Available Cells respond to their environment by modulating protein levels through mRNA transcription and post-transcriptional control. Modest observed correlations between global steady-state mRNA and protein measurements have been interpreted as evidence that mRNA levels determine roughly 40% of the variation in protein levels, indicating dominant post-transcriptional effects. However, the techniques underlying these conclusions, such as correlation and regression, yield biased results when data are noisy, missing systematically, and collinear---properties of mRNA and protein measurements---which motivated us to revisit this subject. Noise-robust analyses of 24 studies of budding yeast reveal that mRNA levels explain more than 85% of the variation in steady-state protein levels. Protein levels are not proportional to mRNA levels, but rise much more rapidly. Regulation of translation suffices to explain this nonlinear effect, revealing post-transcriptional amplification of, rather than competition with, transcriptional signals. These results substantially revise widely credited models of protein-level regulation, and introduce multiple noise-aware approaches essential for proper analysis of many biological phenomena.

  12. Organization and post-transcriptional processing of focal adhesion kinase gene

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    Enslen Hervé

    2006-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase critical for processes ranging from embryo development to cancer progression. Although isoforms with specific molecular and functional properties have been characterized in rodents and chicken, the organization of FAK gene throughout phylogeny and its potential to generate multiple isoforms are not well understood. Here, we study the phylogeny of FAK, the organization of its gene, and its post-transcriptional processing in rodents and human. Results A single orthologue of FAK and the related PYK2 was found in non-vertebrate species. Gene duplication probably occurred in deuterostomes after the echinoderma embranchment, leading to the evolution of PYK2 with distinct properties. The amino acid sequence of FAK and PYK2 is conserved in their functional domains but not in their linker regions, with the absence of autophosphorylation site in C. elegans. Comparison of mouse and human FAK genes revealed the existence of multiple combinations of conserved and non-conserved 5'-untranslated exons in FAK transcripts suggesting a complex regulation of their expression. Four alternatively spliced coding exons (13, 14, 16, and 31, previously described in rodents, are highly conserved in vertebrates. Cis-regulatory elements known to regulate alternative splicing were found in conserved alternative exons of FAK or in the flanking introns. In contrast, other reported human variant exons were restricted to Homo sapiens, and, in some cases, other primates. Several of these non-conserved exons may correspond to transposable elements. The inclusion of conserved alternative exons was examined by RT-PCR in mouse and human brain during development. Inclusion of exons 14 and 16 peaked at the end of embryonic life, whereas inclusion of exon 13 increased steadily until adulthood. Study of various tissues showed that inclusion of these exons also occurred, independently from each other, in a

  13. Control of Candida albicans morphology and pathogenicity by post-transcriptional mechanisms.

    Science.gov (United States)

    Kadosh, David

    2016-11-01

    Candida albicans is a major human fungal pathogen responsible for both systemic and mucosal infections in a wide variety of immunocompromised individuals. Because the ability of C. albicans to undergo a reversible morphological transition from yeast to filaments is important for virulence, significant research efforts have focused on mechanisms that control this transition. While transcriptional and post-translational mechanisms have been well-studied, considerably less is known about the role of post-transcriptional mechanisms. However, in recent years several discoveries have begun to shed light on this important, but understudied, area. Here, I will review a variety of post-transcriptional mechanisms that have recently been shown to control C. albicans morphology, virulence and/or virulence-related processes, including those involving alternative transcript localization, mRNA stability and translation. I will also discuss the role that these mechanisms play in other pathogens as well as the potential they may hold to serve as targets for new antifungal strategies. Ultimately, gaining a better understanding of C. albicans post-transcriptional mechanisms will significantly improve our knowledge of how morphogenesis and virulence are controlled in fungal pathogens and open new avenues for the development of novel and more effective antifungals.

  14. Myc post-transcriptionally induces HIF1 protein and target gene expression in normal and cancer cells

    Science.gov (United States)

    Doe, Megan R.; Ascano, Janice; Kaur, Mandeep; Cole, Michael D.

    2012-01-01

    c-Myc is frequently overexpressed in tumors and plays an important role in the regulation of cancer metabolism. Hypoxia-inducible factor-1 (HIF1), the master regulator of the hypoxic response, enhances tumorigenesis and influences metabolism via upregulation of the glycolytic pathway and suppression of mitochondrial respiration. Together, deregulated Myc and HIF1 cooperate to lend metabolic advantages to proliferating cancer cells and contribute to the Warburg Effect. Here we show that overexpression of Myc significantly stabilizes the alpha subunit of HIF1 (HIF1alpha) under normoxic conditions and enhances HIF1alpha accumulation under hypoxic conditions in cells. Post-transcriptional regulation of HIF1α by Myc led to the induction of HIF1α gene targets. Normoxic HIF1α protein expression was also dependent on Myc. Functionally; HIF1α expression was required for Myc-induced anchorage-independent growth and cell proliferation. Myc-dependent stabilization of HIF1α involved either disruption of binding to the VHL complex or post-translational protein modifications. Taken together, our findings uncover a previously uncharacterized regulatory relationship between Myc and HIF1 that has important implications for cancer metabolism and development. PMID:22186139

  15. A novel post-transcriptional splicing form of the acute T cell leukemia proto-oncogene Lmo2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Lmo2 is a T cell leukemia-related proto-oncogene, which belongs to the LIM protein family. Previous work has established its key role in yolk sac erythropoiesis and adult hematopoiesis, and it is also necessary for regulating angiogenesis. It has been demonstrated that this gene encodes a protein of 158 amino acids, consisting of two tandem cysteine-rich LIM domains, but the detailed mechanism of its transcriptional regulation remains to be elucidated. To further investigate the mechanism of transcriptional regulation of Lmo2, we combined SMART PCR technology with 5′RACE and found a novel post-transcriptional splicing form of Lmo2 in adult human kidney. This alternative transcript contains only two exons, encoding a smaller protein of 151 amino acids. Interestingly, it shares the same reading frame as the original Lmo2, but differs in 7 amino acids at the N-terminus. A genomic DNA fragment (from ?294 nts to +180 nts) containing the putative promoter region has been inserted into the luciferase reporter gene vector pGL3-basic and showed stable promoter activity when transfected into COS7. RT-PCR analysis revealed that this variant transcript was expressed widely in human tissues and cell lines, suggesting its potential basic functional importance.

  16. Post-transcriptional Stabilization of Ucp1 mRNA Protects Mice from Diet-Induced Obesity

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    Akinori Takahashi

    2015-12-01

    Full Text Available Uncoupling protein 1 (Ucp1 contributes to thermogenesis, and its expression is regulated at the transcriptional level. Here, we show that Ucp1 expression is also regulated post-transcriptionally. In inguinal white adipose tissue (iWAT of mice fed a high-fat diet (HFD, Ucp1 level decreases concomitantly with increases in Cnot7 and its interacting partner Tob. HFD-fed mice lacking Cnot7 and Tob express elevated levels of Ucp1 mRNA in iWAT and are resistant to diet-induced obesity. Ucp1 mRNA has an elongated poly(A tail and persists in iWAT of Cnot7−/− and/or Tob−/− mice on a HFD. Ucp1 3′-UTR-containing mRNA is more stable in cells expressing mutant Tob that is unable to bind Cnot7 than in WT Tob-expressing cells. Tob interacts with BRF1, which binds to an AU-rich element in the Ucp1 3′-UTR. BRF1 knockdown partially restores the stability of Ucp1 3′-UTR-containing mRNA. Thus, the Cnot7-Tob-BRF1 axis inhibits Ucp1 expression and contributes to obesity.

  17. Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing

    Science.gov (United States)

    Davuluri, Ganga Rao; van Tuinen, Ageeth; Mustilli, Anna Chiara; Manfredonia, Alessandro; Newman, Robert; Burgess, Diane; Brummell, David A.; King, Stephen R.; Palys, Joe; Uhlig, John; Pennings, Henk M. J.; Bowler, Chris

    2013-01-01

    Summary The tomato HIGH PIGMENT-2 gene encodes an orthologue of the Arabidopsis nuclear protein DE-ETIOLATED 1 (DET1). From genetic analyses it has been proposed that DET1 is a negative regulator of light signal transduction, and recent results indicate that it may control light-regulated gene expression at the level of chromatin remodelling. To gain further understanding about the function of DET1 during plant development, we generated a range of overexpression constructs and introduced them into tomato. Unexpectedly, we only observed phenotypes characteristic of DET1 inactivation, i.e. hyper-responsiveness to light. Molecular analysis indicated in all cases that these phenotypes were a result of suppression of endogenous DET1 expression, due to post-transcriptional gene silencing. DET1 silencing was often lethal when it occurred at relatively early stages of plant development, whereas light hyper-responsive phenotypes were obtained when silencing occurred later on. The appearance of phenotypes correlated with the generation of siRNAs but not DNA hypermethylation, and was most efficient when using constructs with mutations in the DET1 coding sequence or with constructs containing only the 3′-terminal portion of the gene. These results indicate an important function for DET1 throughout plant development and demonstrate that silencing of DET1 in fruits results in increased carotenoids, which may have biotechnological potential. PMID:15469492

  18. miR-155对含SH2区域的肌醇5’磷酸酶1转录后调控在急性髓系白血病发病机制中作用的初步研究%Preliminary study of role of post-transcription regulation on SH2 domain-containing inositol 5'-phosphatase 1 gene expression by miR-155 in the pathogenesis of acute myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    薛华; 赵松颖; 王静; 范丽霞; 化罗明; 罗建民

    2015-01-01

    目的 探讨miR-155对人类含SH2区域的肌醇5’磷酸酶1(SHIP1)的转录后调控在急性髓系白血病(AML)发病机制中的作用.方法 应用反转录聚合酶链反应(RT-PCR)法检测30例AML患者miR-155、SHIP1的mRNA表达水平,选取同年龄健康人骨髓为对照组.人白血病U937细胞转染miR-155类似物后,RT-PCR法检测转染细胞中miR-155、SHIP1的mRNA表达水平.Western blot法检测转染后细胞SHIP1、AKT、pAKT蛋白水平.流式细胞术检测转染后细胞凋亡的变化.结果 30例AML患者中,15例AML-M4及AML-M5患者SHIP1蛋白水平较非AML-M4及AML-M5患者明显降低,而miR-155表达水平相应升高(均P< 0.05).U937细胞转染miR-155后,SHIP1蛋白水平较转染阴性对照组降低(P<0.05),而p-AKT水平较转染阴性对照组明显升高,转染后细胞凋亡明显受抑(P<0.05).结论 miR-155可对SHIP1进行转录后调控,miR-155可能通过降低SHIP1活性而激活PI3K-AKT途径,抑制白血病细胞的凋亡,从而促进AML的发生.%Objective To investigate the role of microRNA-155 (miR-155) on post-transcription regulation of SH2 domain-containing inositol 5'-phosphatase 1 (SHIP1) gene expression in the pathogenesis of acute myeloid leukemia (AML).Methods Quantitative real-time polymerase chain reaction (RT-PCR) was performed to detect the expression of miR-155 and SHIP1 mRNA in the AML patients and controls.miR-155 mimics was transfected into U937cells (U937m) by using X-treme GENE siRNA transfection reagent.Cells without transfection (U937c) and cells with negative transfection (U937mc) were used as controls.RT-PCR was performed to detect the expression of miR-155 and SHIP1 mRNA in these cells.The expression of SHIP1,TAKT and pAKT were detected by Western blot in U937 cells.Apoptosis was studied by flow cytometry (FCM).Results The average level of SHIP1 protein content in 15 samples of patients with AML-M4 or AML-M5 from 30 AML patients was significantly lower compared with that of

  19. Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development.

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    Gunnar R Mair

    2010-02-01

    Full Text Available A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch. This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.

  20. Localizing potentially active post-transcriptional regulations in the Ewing's sarcoma gene regulatory network

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    Delyon Bernard

    2010-11-01

    Full Text Available Abstract Background A wide range of techniques is now available for analyzing regulatory networks. Nonetheless, most of these techniques fail to interpret large-scale transcriptional data at the post-translational level. Results We address the question of using large-scale transcriptomic observation of a system perturbation to analyze a regulatory network which contained several types of interactions - transcriptional and post-translational. Our method consisted of post-processing the outputs of an open-source tool named BioQuali - an automatic constraint-based analysis mimicking biologist's local reasoning on a large scale. The post-processing relied on differences in the behavior of the transcriptional and post-translational levels in the network. As a case study, we analyzed a network representation of the genes and proteins controlled by an oncogene in the context of Ewing's sarcoma. The analysis allowed us to pinpoint active interactions specific to this cancer. We also identified the parts of the network which were incomplete and should be submitted for further investigation. Conclusions The proposed approach is effective for the qualitative analysis of cancer networks. It allows the integrative use of experimental data of various types in order to identify the specific information that should be considered a priority in the initial - and possibly very large - experimental dataset. Iteratively, new dataset can be introduced into the analysis to improve the network representation and make it more specific.

  1. The exon junction complex as a node of post-transcriptional networks.

    Science.gov (United States)

    Le Hir, Hervé; Saulière, Jérôme; Wang, Zhen

    2016-01-01

    The exon junction complex (EJC) is deposited onto mRNAs following splicing and adopts a unique structure, which can both stably bind to mRNAs and function as an anchor for diverse processing factors. Recent findings revealed that in addition to its established roles in nonsense-mediated mRNA decay, the EJC is involved in mRNA splicing, transport and translation. While structural studies have shed light on EJC assembly, transcriptome-wide analyses revealed differential EJC loading at spliced junctions. Thus, the EJC functions as a node of post-transcriptional gene expression networks, the importance of which is being revealed by the discovery of increasing numbers of EJC-related disorders.

  2. Cyclosporin A Decreases Human Macrophage Interleukin-6 Synthesis at Post-Transcriptional Level

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    Juan E. Losa García

    1999-01-01

    Full Text Available In addition to its well-established effect on T cells, cyclosporin A (CsA also inhibits inflammatory cytokine production by macrophages. However, little is known about the mechanism of action of CsA on macrophage cytokine production. We measured the effect of CsA on basal and phorbol-myristate-acetate (PMA-stimulated production of interleukin-6 using the human monocyte cell line U937 differentiated with dimethylsulfoxide (DMSO. Interleukin-6 levels were measured in supernatant and cell lysates using specific enzyme-linked immunosorbent assays. We found that CsA decreases not only IL-6 release but also cytokine synthesis. The concentration of CsA used did not affect either cell viability or proliferation. Three possibilities may be advanced to explain the CsA-due decrease in IL-6 production by macrophages: (a inhibition of the synthesis of an early common regulatory protein, (b inhibition of cytokine gene transcription, or (c modulation of post-transcriptional events. The first possibility was tested by measuring the effect of cycloheximide on the experimental system during the first 3 hours of culture. Although cycloheximide decreased total cytokine synthesis, the pattern of cytokine modulation by CsA persisted. These data suggest that CsA-mediated macrophage cytokine inhibition is not mediated by an early common regulatory protein. To further explore the inhibition mechanism, we measured IL-6 mRNA levels by Northern blot. IL-6 mRNA levels were unaffected by CsA both in resting and PMA-stimulated cells. We conclude that in human macrophages CsA diminishes IL-6 production at post-transcriptional level.

  3. Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set.

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    Monika Mahajan

    Full Text Available Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi through post-transcriptional gene silencing (PTGS of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25-93% reduction in flavonol (quercetin content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless

  4. Preliminary identification and analysis of point mutations corre-lated with response to interferon-α in hepatitis B virus post-transcriptional regulatory elements

    Institute of Scientific and Technical Information of China (English)

    XING Tong-jing; LUO Kang-xian; HOU Jin-lin

    2005-01-01

    Background It is still unclear whether viral genetic variability influences response to interferon(IFN)-α treatment. Recent reports suggest that IFN-α effects may be associated with hepatitis B virus(HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-α treatment. Methods The HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines. Results The T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-α/γ and tumor necrosis factor (TNF)-α except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-α on HPRE. Conclusions There are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-α. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-α other than that of IFN-γ or TNF-α on HPRE.

  5. Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

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    Jenny M McLaughlan

    Full Text Available The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L, changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae.

  6. Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti.

    Science.gov (United States)

    Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

    2014-01-01

    The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σ(E2) or σ(H1/2). Recovery rates of sRNAs in each of the CoIP-RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σ(E2)-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5' regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA-mRNA regulatory pairs.

  7. The cystic-fibrosis-associated ΔF508 mutation confers post-transcriptional destabilization on the C. elegans ABC transporter PGP-3

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    Liping He

    2012-11-01

    Membrane proteins make up ∼30% of the proteome. During the early stages of maturation, this class of proteins can experience localized misfolding in distinct cellular compartments, such as the cytoplasm, endoplasmic reticulum (ER lumen and ER membrane. ER quality control (ERQC mechanisms monitor folding and determine whether a membrane protein is appropriately folded or is misfolded and warrants degradation. ERQC plays crucial roles in human diseases, such as cystic fibrosis, in which deletion of a single amino acid (F508 results in the misfolding and degradation of the cystic fibrosis transmembrane conductance regulator (CFTR Cl– channel. We introduced the ΔF508 mutation into Caenorhabditis elegans PGP-3, a 12-transmembrane ABC transporter with 15% identity to CFTR. When expressed in intestinal epithelial cells, PGP-3wt was stable and efficiently trafficked to the apical plasma membrane through a COPII-dependent mechanism. However, PGP-3ΔF508 was post-transcriptionally destabilized, resulting in reduced total and apical membrane protein levels. Genetic or physiological activation of the osmotic stress response pathway, which causes accumulation of the chemical chaperone glycerol, stabilized PGP-3ΔF508. Efficient degradation of PGP-3ΔF508 required the function of several C. elegans ER-associated degradation (ERAD homologs, suggesting that destabilization occurs through an ERAD-type mechanism. Our studies show that the ΔF508 mutation causes post-transcriptional destabilization and degradation of PGP-3 in C. elegans epithelial cells. This model, combined with the power of C. elegans genetics, provides a new opportunity to genetically dissect metazoan ERQC.

  8. Evolutionary Profiling of the Transcriptional and Post-Transcriptional Wiring of the Circadian Clock in Normal and Perturbed Conditions

    Science.gov (United States)

    2014-08-14

    how the circadian cycle adapts to environmental stress. More specifically: As expected, Drosophila melanogaster showed two peaks of activity: one in...transcriptional and post-transcriptional wiring of the circadian clock in normal and perturbed conditions Contract Number: DARPA D13AP00074 Total...out behavioral analysis of the different fly species and their response to temperature changes. Towards this, we tested the circadian locomotor

  9. Distinct regulatory mechanisms of the human ferritin gene by hypoxia and hypoxia mimetic cobalt chloride at the transcriptional and post-transcriptional levels.

    Science.gov (United States)

    Huang, Bo-Wen; Miyazawa, Masaki; Tsuji, Yoshiaki

    2014-12-01

    Cobalt chloride has been used as a hypoxia mimetic because it stabilizes hypoxia inducible factor-1α (HIF1-α) and activates gene transcription through a hypoxia responsive element (HRE). However, differences between hypoxia and hypoxia mimetic cobalt chloride in gene regulation remain elusive. Expression of ferritin, the major iron storage protein, is regulated at the transcriptional and posttranscriptional levels through DNA and RNA regulatory elements. Here we demonstrate that hypoxia and cobalt chloride regulate ferritin heavy chain (ferritin H) expression by two distinct mechanisms. Both hypoxia and cobalt chloride increased HIF1-α but a putative HRE in the human ferritin H gene was not activated. Instead, cobalt chloride but not hypoxia activated ferritin H transcription through an antioxidant responsive element (ARE), to which Nrf2 was recruited. Intriguingly, cobalt chloride downregulated ferritin H protein expression while it upregulated other ARE-regulated antioxidant genes in K562 cells. Further characterization demonstrated that cobalt chloride increased interaction between iron regulatory proteins (IRP1 and IRP2) and iron responsive element (IRE) in the 5'UTR of ferritin H mRNA, resulting in translational block of the accumulated ferritin H mRNA. In contrast, hypoxia had marginal effect on ferritin H transcription but increased its translation through decreased IRP1-IRE interaction. These results suggest that hypoxia and hypoxia mimetic cobalt chloride employ distinct regulatory mechanisms through the interplay between DNA and mRNA elements at the transcriptional and post-transcriptional levels.

  10. Identification of novel microRNAs in post-transcriptional control of Nrf2 expression and redox homeostasis in neuronal, SH-SY5Y cells.

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    Madhusudhanan Narasimhan

    Full Text Available Nuclear factor-erythroid 2-related factor 2 (Nrf2/NFE2L2, a redox-sensitive transcription factor plays a critical role in adaptation to cellular stress and affords cellular defense by initiating transcription of antioxidative and detoxification genes. While a protein can be regulated at multiple levels, control of Nrf2 has been largely studied at post-translational regulation points by Keap1. Importantly, post-transcriptional/translational based regulation of Nrf2 is less understood and to date there are no reports on such mechanisms in neuronal systems. In this context, studies involving the role of microRNAs (miRs which are normally considered as fine tuning regulators of protein production through translation repression and/or post-transcriptional alterations, are in place. In the current study, based on in-silico analysis followed by immunoblotting and real time analysis, we have identified and validated for the first time that human NFE2L2 could be targeted by miR153/miR27a/miR142-5p/miR144 in neuronal, SH-SY5Y cells. Co-transfection studies with individual miR mimics along with either WT 3' UTR of human Nrf2 or mutated miRNA targeting seed sequence within Nrf2 3' UTR, demonstrated that Nrf2 is a direct regulatory target of these miRs. In addition, ectopic expression of miR153/miR27a/miR142-5p/miR144 affected Nrf2 mRNA abundance and nucleo-cytoplasmic concentration of Nrf2 in a Keap1 independent manner resulting in inefficient transactivating ability of Nrf2. Furthermore, forced expression of miRs diminished GCLC and GSR expression resulting in alteration of Nrf2 dependent redox homeostasis. Finally, bioinformatics based miRNA-disease network analysis (MDN along with extended computational network analysis of Nrf2 associated pathologic processes suggests that if in a particular cellular scenario where any of these miR153/miR27a/miR142-5p/miR144 either individually or as a group is altered, it could affect Nrf2 thus triggering and

  11. Efficient use of artificial micro-RNA to downregulate the expression of genes at the post-transcriptional level in Arabidopsis thaliana.

    Science.gov (United States)

    Ud-Din, A; Rauf, M; Ghafoor, S; Khattak, M N K; Hameed, M W; Shah, H; Jan, S; Muhammad, K; Rehman, A; Inamullah

    2016-04-07

    Micro-RNAs are cellular components regulating gene expression at the post-transcription level. In the present study, artificial micro-RNAs were used to decrease the transcript level of two genes, AtExpA8 (encoding an expansin) and AHL25 (encoding an AT-hook motif nuclear localized protein) in Arabidopsis thaliana. The backbone of the Arabidopsis endogenous MIR319a micro-RNA was used in a site-directed mutagenesis approach for the generation of artificial micro-RNAs targeting two genes. The recombinant cassettes were expressed under the control of the CaMV 35S promoter in individual A. thaliana plants. Transgenic lines of the third generation were tested by isolating total RNA and by subsequent cDNA synthesis using oligo-dT18 primers and mRNAs as templates. The expression of the two target genes was checked through quantitative real-time polymerase chain reaction to confirm reduced transcript levels for AtExpA8 and AHL25. Downregulation of AtExpA8 resulted in the formation of short hypocotyls compared with those of the wild-type control in response to low pH and high salt concentration. This technology could be used to prevent the expression of exogenous and invading genes posing a threat to the normal cellular physiology of the host plant.

  12. Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells

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    Canto Tomas

    2004-08-01

    Full Text Available Abstract Background RNA interference (RNAi in animals and post-transcriptional gene silencing (PTGS in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. Results Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. Conclusion These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.

  13. PARP1 promotes gene expression at the post-transcriptional level by modulating the RNA-binding protein HuR

    Science.gov (United States)

    Ke, Yueshuang; Han, Yanlong; Guo, Xiaolan; Wen, Jitao; Wang, Ke; Jiang, Xue; Tian, Xue; Ba, Xueqing; Boldogh, Istvan; Zeng, Xianlu

    2017-01-01

    Poly(ADP-ribosyl)ation (PARylation) is mainly catalysed by poly-ADP-ribose polymerase 1 (PARP1), whose role in gene transcription modulation has been well established. Here we show that, in response to LPS exposure, PARP1 interacts with the adenylateuridylate-rich element-binding protein embryonic lethal abnormal vision-like 1 (Elavl1)/human antigen R (HuR), resulting in its PARylation, primarily at site D226. PARP inhibition and the D226 mutation impair HuR's PARylation, nucleocytoplasmic shuttling and mRNA binding. Increases in mRNA level or stability of pro-inflammatory cytokines/chemokines are abolished by PARP1 ablation or inhibition, or blocked in D226A HuR-expressing cells. The present study demonstrates a mechanism to regulate gene expression at the post-transcriptional level, and suggests that blocking the interaction of PARP1 with HuR could be a strategy to treat inflammation-related diseases that involve increased mRNA stability. PMID:28272405

  14. Role of a redox-based methylation switch in mRNA life cycle (pre- and post-transcriptional maturation) and protein turnover: implications in neurological disorders.

    Science.gov (United States)

    Trivedi, Malav S; Deth, Richard C

    2012-01-01

    Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation, and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition, and behavior (Cajigas et al., 2010). Thus a regulatory switch, which controls the lifespan, maturation, and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at (1) the pre-transcription level, by regulating precursor-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and (2) the post-transcription level by modulating the regulatory functions of ribonucleoproteins and RNA binding proteins in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of

  15. Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

    Directory of Open Access Journals (Sweden)

    MALAV SUCHIN TRIVEDI

    2012-06-01

    Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

  16. Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2005-03-01

    Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

  17. Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

    Directory of Open Access Journals (Sweden)

    Miller Eric S

    2010-12-01

    Full Text Available Abstract Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit; and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.

  18. Post-transcriptional control of DGCR8 expression by the Microprocessor.

    Science.gov (United States)

    Triboulet, Robinson; Chang, Hao-Ming; Lapierre, Robert J; Gregory, Richard I

    2009-06-01

    The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.

  19. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq

    DEFF Research Database (Denmark)

    Sittka, A; Lucchini, S; Papenfort, K;

    2008-01-01

    would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high...

  20. Diverse mechanisms of post-transcriptional repression by the small RNA regulator of glucose-phosphate stress.

    Science.gov (United States)

    Bobrovskyy, Maksym; Vanderpool, Carin K

    2016-01-01

    The Escherichia coli small RNA SgrS controls a metabolic stress response that occurs upon accumulation of certain glycolytic intermediates. SgrS base pairs with and represses translation of ptsG and manXYZ mRNAs, which encode sugar transporters, and activates translation of yigL mRNA, encoding a sugar phosphatase. This study defines four new genes as direct targets of E. coli SgrS. These new targets, asd, adiY, folE and purR, encode transcription factors or enzymes of diverse metabolic pathways, including aspartate semialdehyde dehydrogenase, arginine decarboxylase gene activator, GTP cyclohydrolase I and a repressor of purine biosynthesis, respectively. SgrS represses translation of each of the four target mRNAs via distinct mechanisms. SgrS binding sites overlapping the Shine-Dalgarno sequences of adiY and folE mRNAs suggest that SgrS pairing with these targets directly occludes ribosome binding and prevents translation initiation. SgrS binding within the purR coding sequence recruits the RNA chaperone Hfq to directly repress purR translation. Two separate SgrS binding sites were found on asd mRNA, and both are required for full translational repression. Ectopic overexpression of asd, adiY and folE is specifically detrimental to cells experiencing glucose-phosphate stress, suggesting that SgrS-dependent repression of the metabolic functions encoded by these targets promotes recovery from glucose-phosphate stress.

  1. The bZip transcription factor vitellogenin-binding protein is post transcriptional down regulated in chicken liver

    NARCIS (Netherlands)

    Smidt, MP; Snippe, L; Van Keulen, G; Ab, G

    1998-01-01

    The vitellogenin-binding protein (VBP) is a member of the proline and acidic-region rich (PAR) family of bZip transcription factors. PAR is located N-terminally to the DNA-binding domain. VBP binds to specific sites within the 300-bp 5'-flanking region of the chicken-liver-specific estrogen-dependen

  2. Members of a large retroposon family are determinants of post-transcriptional gene expression in Leishmania.

    Directory of Open Access Journals (Sweden)

    Frédéric Bringaud

    2007-09-01

    Full Text Available Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis, Trypanosoma brucei (sleeping sickness, and Trypanosoma cruzi (Chagas disease. Analysis of their recently completed genomes confirmed the presence of non-long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements--LmSIDER1 (785 copies and LmSIDER2 (1,073 copies--that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are approximately 70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3'-untranslated regions (3'UTRs of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3'UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.

  3. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

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    Ziga Zebec

    2016-10-01

    Full Text Available CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.

  4. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    Science.gov (United States)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  5. A conserved region in the 3' untranslated region of the human LIMK1 gene is critical for proper expression of LIMK1 at the post-transcriptional level

    Institute of Scientific and Technical Information of China (English)

    Guang-Fei Deng; Shu-Jing Liu; Xun-Sha Sun; Wei-Wen Sun; Qi-Hua Zhao; Wei-Ping Liao; Yong-Hong Yi

    2013-01-01

    LIM kinase 1 (LIMK1),a cytosolic serine/threonine kinase,regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function.Abnormal expression of LIMK1 is associated with several neurological disorders.In this study,we performed a conservation analysis using Vector NTI (8.0) software.The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene,respectively.We found that a region ranging from nt +884 to +966 in the human LIMK1 3' untranslated region (UTR) was highly conserved in the mouse Limk1 3' UTR and formed a structure containing several loops and stems.Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted,pGL4-hLIMK1-3U-M,in SH-SY5Y and HEK-293 cells was only ~60% of that of the wild-type construct pGL4-hLIMK1-3U,indicating that the conserved region is critical for the reporter gene expression.Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to ~50% of that in cells transfected with pGL4-hLIMK1-3U,suggesting an important role of the conserved region in maintaining Luc2 mRNA stability.Our study suggests that the conserved region in the LIMK1 3' UTR is involved in regulating LIMK1 expression at the post-transcriptional level,which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3'-UTR mutant and neurological disorders.

  6. Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4

    Directory of Open Access Journals (Sweden)

    Wise Petra M

    2010-04-01

    Full Text Available Abstract Background Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E2 is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4, an enzyme that converts E2 to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ and females (ZW because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E2 in their brains than females. Results Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain. Conclusions Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E2 metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms.

  7. Optimisation of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes

    Directory of Open Access Journals (Sweden)

    Defez Roberto

    2002-01-01

    Full Text Available Abstract Background Genetic engineering of parthenocarpy confers to horticultural plants the ability to produce fruits under environmental conditions that curtail fruit productivity and quality. The DefH9-iaaM transgene, whose predicted action is to confer auxin synthesis specifically in the placenta, ovules and derived tissues, has been shown to confer parthenocarpy to several plant species (tobacco, eggplant, tomato and varieties. Results UC82 tomato plants, a typical cultivar used by the processing industry, transgenic for the DefH9-iaaM gene produce parthenocarpic fruits that are malformed. UC82 plants transgenic for the DefH9-RI-iaaM, a DefH9-iaaM derivative gene modified in its 5'ULR by replacing 53 nucleotides immediately upstream of the AUG initiation codon with an 87 nucleotides-long sequence derived from the rolA intron sequence, produce parthenocarpic fruits of high quality. In an in vitro translation system, the iaaM mRNA, modified in its 5'ULR is translated 3–4 times less efficiently than the original transcript. An optimal expressivity of parthenocarpy correlates with a reduced transgene mRNA steady state level in DefH9-RI-iaaM flower buds in comparison to DefH9-iaaM flower buds. Consistent with the known function of the iaaM gene, flower buds transgenic for the DefH9-RI-iaaM gene contain ten times more IAA than control untransformed flower buds, but five times less than DefH9-iaaM flower buds. Conclusions By using an auxin biosynthesis transgene downregulated at the post-transcriptional level, an optimal expressivity of parthenocarpy has been achieved in a genetic background not suitable for the original transgene. Thus, the method allows the generation of a wider range of expressivity of the desired trait in transgenic plants.

  8. 3-(3-amino-3-carboxypropyl)-5,6-Dihydrouridine is one of two novel post-transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Krog, Jesper Schak; Español, Yaiza; Giessing, Anders M B;

    2011-01-01

    was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence......tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar....... Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys) (UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step...

  9. T cell post-transcriptional miRNA-mRNA interaction networks identify targets associated with susceptibility/resistance to collagen-induced arthritis.

    Directory of Open Access Journals (Sweden)

    Paula B Donate

    Full Text Available BACKGROUND: Due to recent studies indicating that the deregulation of microRNAs (miRNAs in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. METHODOLOGY/PRINCIPAL FINDINGS: To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+ T cells from two mouse strains the DBA-1/J strain (MHC-H2q, which is susceptible to collagen induced arthritis (CIA, and the DBA-2/J strain (MHC-H2d, which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++ algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt. FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. CONCLUSIONS/SIGNIFICANCE: This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.

  10. Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

    Science.gov (United States)

    Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

    2016-02-01

    Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

  11. Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

    Science.gov (United States)

    Ohno, Sho; Hosokawa, Munetaka; Kojima, Misa; Kitamura, Yoshikuni; Hoshino, Atsushi; Tatsuzawa, Fumi; Doi, Motoaki; Yazawa, Susumu

    2011-11-01

    Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

  12. The Csr/Rsm system of Yersinia and related pathogens: a post-transcriptional strategy for managing virulence.

    Science.gov (United States)

    Heroven, Ann Kathrin; Böhme, Katja; Dersch, Petra

    2012-04-01

    This review emphasizes the function and regulation of the Csr regulatory system in the human enteropathogen Yersinia pseudotuberculosis and compares its features with the homologous Csr/Rsm systems of related pathogens. The Csr/Rsm systems of eubacteria form a complex regulatory network in which redundant non-translated Csr/Rsm-RNAs bind the RNA-binding protein CsrA/RsmA, thereby preventing its interaction with mRNA targets. The Csr system is controlled by the BarA/UvrY-type of two-component sensor-regulator systems. Apart from that, common or pathogen-specific regulators control the abundance of the Csr components. The coordinate control of virulence factors and infection-linked physiological traits by the Csr/Rsm systems helps the pathogens to adapt individually to rapidly changing conditions to which they are exposed during the different stages of an infection. As Csr/Rsm function is relevant for full virulence, it represents a target suitable for antimicrobial drug development.

  13. MicroRNAs, macrocontrol : Regulation of miRNA processing

    NARCIS (Netherlands)

    Slezak-Prochazka, Izabella; Durmus, Selvi; Kroesen, Bart-Jan; van den Berg, Anke

    2010-01-01

    MicroRNAs (miRNAs) are a set of small, non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. Maturation of miRNAs comprises several regulated steps resulting in similar to 22-nucleotide single-stranded mature miRNAs. Regulation of miRNA expression can occur both at

  14. The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

    Science.gov (United States)

    Vasseur, Romain; Skrypek, Nicolas; Duchêne, Belinda; Renaud, Florence; Martínez-Maqueda, Daniel; Vincent, Audrey; Porchet, Nicole; Van Seuningen, Isabelle; Jonckheere, Nicolas

    2015-12-01

    The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

  15. A Systems Biology Overview on Human Diabetic Nephropathy: From Genetic Susceptibility to Post-Transcriptional and Post-Translational Modifications.

    Science.gov (United States)

    Conserva, Francesca; Gesualdo, Loreto; Papale, Massimo

    2016-01-01

    Diabetic nephropathy (DN), a microvascular complication occurring in approximately 20-40% of patients with type 2 diabetes mellitus (T2DM), is characterized by the progressive impairment of glomerular filtration and the development of Kimmelstiel-Wilson lesions leading to end-stage renal failure (ESRD). The causes and molecular mechanisms mediating the onset of T2DM chronic complications are yet sketchy and it is not clear why disease progression occurs only in some patients. We performed a systematic analysis of the most relevant studies investigating genetic susceptibility and specific transcriptomic, epigenetic, proteomic, and metabolomic patterns in order to summarize the most significant traits associated with the disease onset and progression. The picture that emerges is complex and fascinating as it includes the regulation/dysregulation of numerous biological processes, converging toward the activation of inflammatory processes, oxidative stress, remodeling of cellular function and morphology, and disturbance of metabolic pathways. The growing interest in the characterization of protein post-translational modifications and the importance of handling large datasets using a systems biology approach are also discussed.

  16. A Systems Biology Overview on Human Diabetic Nephropathy: From Genetic Susceptibility to Post-Transcriptional and Post-Translational Modifications

    Directory of Open Access Journals (Sweden)

    Francesca Conserva

    2016-01-01

    Full Text Available Diabetic nephropathy (DN, a microvascular complication occurring in approximately 20–40% of patients with type 2 diabetes mellitus (T2DM, is characterized by the progressive impairment of glomerular filtration and the development of Kimmelstiel-Wilson lesions leading to end-stage renal failure (ESRD. The causes and molecular mechanisms mediating the onset of T2DM chronic complications are yet sketchy and it is not clear why disease progression occurs only in some patients. We performed a systematic analysis of the most relevant studies investigating genetic susceptibility and specific transcriptomic, epigenetic, proteomic, and metabolomic patterns in order to summarize the most significant traits associated with the disease onset and progression. The picture that emerges is complex and fascinating as it includes the regulation/dysregulation of numerous biological processes, converging toward the activation of inflammatory processes, oxidative stress, remodeling of cellular function and morphology, and disturbance of metabolic pathways. The growing interest in the characterization of protein post-translational modifications and the importance of handling large datasets using a systems biology approach are also discussed.

  17. Identifying Aspects of the Post-Transcriptional Program Governing the Proteome of the Green Alga Micromonas pusilla

    Energy Technology Data Exchange (ETDEWEB)

    Waltman, Peter H.; Guo, Jian; Reistetter, Emily Nahas; Purvine, Samuel; Ansong, Charles K.; van Baren, Marijke J.; Wong, Chee-Hong; Wei, Chia-Lin; Smith, Richard D.; Callister, Stephen J.; Stuart, Joshua M.; Worden, Alexandra Z.; Mills, Ken

    2016-07-19

    Micromonas is a unicellular green alga that belongs to the prasinophytes, a sister lineage to land plants. This picoeukaryotic (<2 μm diameter) alga is widespread in the marine environment but still not understood at the cellular level. Here, we examine the mRNA and protein level changes that take place over the course of the day-night cycle using mid-exponential nutrient replete cultures of Micromonas pusilla CCMP1545 grown and analyzed in biological triplicate. During the experiment, samples were collected at key transition points during the diel for evaluation using high-throughput LC-MS proteomics. We also sequenced matched mRNA samples from the same time points, using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels using both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including the codon usage and the length of the 3’ UTR. Collectively, our studies provide insights into the regulation of the proteome over a diel as relationships between the transcriptional and translational programs in the widespread marine green alga Micromonas.

  18. Identifying Aspects of the Post-Transcriptional Program Governing the Proteome of the Green Alga Micromonas pusilla.

    Directory of Open Access Journals (Sweden)

    Peter H Waltman

    Full Text Available Micromonas is a unicellular motile alga within the Prasinophyceae, a green algal group that is related to land plants. This picoeukaryote (<2 μm diameter is widespread in the marine environment but is not well understood at the cellular level. Here, we examine shifts in mRNA and protein expression over the course of the day-night cycle using triplicated mid-exponential, nutrient replete cultures of Micromonas pusilla CCMP1545. Samples were collected at key transition points during the diel cycle for evaluation using high-throughput LC-MS proteomics. In conjunction, matched mRNA samples from the same time points were sequenced using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as we observed in the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels from both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including codon usage as well as 3' UTR length and structure. Collectively, our studies provide insights into the regulation of the proteome over a diel cycle as well as the relationships between transcriptional and translational programs in the widespread marine green alga Micromonas.

  19. Identifying Aspects of the Post-Transcriptional Program Governing the Proteome of the Green Alga Micromonas pusilla.

    Science.gov (United States)

    Waltman, Peter H; Guo, Jian; Reistetter, Emily Nahas; Purvine, Samuel; Ansong, Charles K; van Baren, Marijke J; Wong, Chee-Hong; Wei, Chia-Lin; Smith, Richard D; Callister, Stephen J; Stuart, Joshua M; Worden, Alexandra Z

    2016-01-01

    Micromonas is a unicellular motile alga within the Prasinophyceae, a green algal group that is related to land plants. This picoeukaryote (<2 μm diameter) is widespread in the marine environment but is not well understood at the cellular level. Here, we examine shifts in mRNA and protein expression over the course of the day-night cycle using triplicated mid-exponential, nutrient replete cultures of Micromonas pusilla CCMP1545. Samples were collected at key transition points during the diel cycle for evaluation using high-throughput LC-MS proteomics. In conjunction, matched mRNA samples from the same time points were sequenced using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as we observed in the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels from both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including codon usage as well as 3' UTR length and structure. Collectively, our studies provide insights into the regulation of the proteome over a diel cycle as well as the relationships between transcriptional and translational programs in the widespread marine green alga Micromonas.

  20. MicroRNA Regulation of SIRT1

    OpenAIRE

    Yamakuchi, Munekazu

    2012-01-01

    SIRT1 is an NAD-dependent deacetylase that regulates stress response pathways. By deacetylating transcription factors and co-factors, SIRT1 modulates metabolism, inflammation, hypoxic responses, circadian rhythms, cell survival, and longevity. Since SIRT1 plays a key role in regulating pathways involved in cardiovascular diseases and metabolic diseases cancer, the regulation of SIRT1 has received intense scrutiny. The post-transcriptional regulation of SIRT1 is mediated by two classes of mole...

  1. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    Science.gov (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

  2. MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, S.Y. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Radiation-Oncology; HungKuang Univ., Taichung (China). Dept. of Biotechnology; Lin, K.C. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Oral and Maxillofacial Surgery; Chiou, J.F. [Taipei Medical Univ., Taipei (China). Dept. of Radiology; Taipei Medical Univ. Hospital, Taipei (China). Dept. of Radiation Oncology; Taipei Medical Univ. Hospital, Taipei (China). Dept. of Hospice and Palliative Center; Taipei Medical Univ. Hospital, Taipei (China). Cancer Center; Jeng, S.C. [Taipei Medical Univ. Hospital, Taipei (China). Dept. of Radiation Oncology; Cheng, W.H.; Chang, C.I. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Hemato-Ongology; Lin, W.C. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Div. of Thoracic Surgery; Wu, L.L. [National Taiwan Univ. Hospital, Taipei (China). Dept. of Ophthalmology; Lee, H.L. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Radiation-Oncology; Chen, R.J. [National Taiwan Univ. Hospital and National Taiwan Univ., Taipei (China). Dept. of Obstetrics and Gynecology

    2013-08-15

    Background and purpose: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. Materials and methods: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. Results: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. Conclusion: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells. (orig.)

  3. p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

    Directory of Open Access Journals (Sweden)

    Eileen M McGowan

    Full Text Available As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation, and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

  4. Mungbean yellow mosaic virus (MYMV) AC4 suppresses post-transcriptional gene silencing and an AC4 hairpin RNA gene reduces MYMV DNA accumulation in transgenic tobacco.

    Science.gov (United States)

    Sunitha, Sukumaran; Shanmugapriya, Gnanasekaran; Balamani, Veluthambi; Veluthambi, Karuppannan

    2013-06-01

    Mungbean yellow mosaic virus (MYMV) is a legume-infecting geminivirus that causes yellow mosaic disease in blackgram, mungbean, soybean, Frenchbean and mothbean. AC4/C4, which is nested completely within the Rep gene, is less conserved among geminiviruses. Much less is known about its role in viral pathogenesis other than its known role in the suppression of host-mediated gene silencing. Transient expression of MYMV AC4 by agroinfiltration suppressed post-transcriptional gene silencing in Nicotiana benthamiana 16c expressing green fluorescence protein, at a level comparable to MYMV TrAP expression. AC4 full-length gene and an inverted repeat of AC4 (comprising the full-length AC4 sequence in sense and antisense orientations with an intervening intron) which makes a hairpin RNA (hpRNA) upon transcription were introduced into tobacco by Agrobacterium-mediated leaf disc transformation. Leaf discs of the transgenic plants were agroinoculated with partial dimers of MYMV and used to study the effect of the AC4-sense and AC4 hpRNA genes on MYMV DNA accumulation. Leaf discs of two transgenic plants that express the AC4-sense gene displayed an increase in MYMV DNA accumulation. Leaf discs of six transgenic plants containing the AC4 hpRNA gene accumulated small-interfering RNAs (siRNAs) specific to AC4, and upon agroinoculation with MYMV they exhibited a severe reduction in the accumulation of MYMV DNA. Thus, the MYMV AC4 hpRNA gene has emerged as a good candidate to engineer resistance against MYMV in susceptible plants.

  5. Transcriptional and Post-Transcriptional Modulation of SPI1 and SPI2 Expression by ppGpp, RpoS and DksA in Salmonella enterica sv Typhimurium.

    Science.gov (United States)

    Rice, Christopher J; Ramachandran, Vinoy K; Shearer, Neil; Thompson, Arthur

    2015-01-01

    The expression of genes within Salmonella Pathogenicity Islands 1 and 2 (SPI1, SPI2) is required to facilitate invasion and intracellular replication respectively of S. Typhimurium in host cell lines. Control of their expression is complex and occurs via a variety of factors operating at transcriptional and post-transcriptional levels in response to the environmental stimuli found within the host. Several of the factors that modulate SPI1 and SPI2 expression are involved in the redistribution or modification of RNA polymerase (RNAP) specificity. These factors include the bacterial alarmone, ppGpp, the alternative sigma factor, RpoS, and the RNAP accessory protein, DksA. In this report we show not only how these three factors modulate SPI1 and SPI2 expression but also how they contribute to the 'phased' expression of SPI1 and SPI2 during progress through late-log and stationary phase in aerobic rich broth culture conditions. In addition, we demonstrate that the expression of at least one SPI1-encoded protein, SipC is subject to DksA-dependent post-transcriptional control.

  6. Transcriptional and Post-Transcriptional Modulation of SPI1 and SPI2 Expression by ppGpp, RpoS and DksA in Salmonella enterica sv Typhimurium.

    Directory of Open Access Journals (Sweden)

    Christopher J Rice

    Full Text Available The expression of genes within Salmonella Pathogenicity Islands 1 and 2 (SPI1, SPI2 is required to facilitate invasion and intracellular replication respectively of S. Typhimurium in host cell lines. Control of their expression is complex and occurs via a variety of factors operating at transcriptional and post-transcriptional levels in response to the environmental stimuli found within the host. Several of the factors that modulate SPI1 and SPI2 expression are involved in the redistribution or modification of RNA polymerase (RNAP specificity. These factors include the bacterial alarmone, ppGpp, the alternative sigma factor, RpoS, and the RNAP accessory protein, DksA. In this report we show not only how these three factors modulate SPI1 and SPI2 expression but also how they contribute to the 'phased' expression of SPI1 and SPI2 during progress through late-log and stationary phase in aerobic rich broth culture conditions. In addition, we demonstrate that the expression of at least one SPI1-encoded protein, SipC is subject to DksA-dependent post-transcriptional control.

  7. Towards an understanding of miRNA regulation

    DEFF Research Database (Denmark)

    Jensen, Trine Ilsø

    2015-01-01

    miRNAs are well-known regulators of gene expression. They function post-transcriptionally by binding to complementary sites within the 3´UTR of target mRNAs, which mediates translational repression and destabilization. However, miRNA expression itself is also subjected to regulation. Here, we rep...... and transportation within the cell was pursued by investigating the use of a MS2 system to accommodate both project aims. We inserted MS2 stem-loops into ciRS-7 and preliminary results of ciRS-7 immuno-precipitation and ciRS-7 localization in the cell are presented.......miRNAs are well-known regulators of gene expression. They function post-transcriptionally by binding to complementary sites within the 3´UTR of target mRNAs, which mediates translational repression and destabilization. However, miRNA expression itself is also subjected to regulation. Here, we...

  8. Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells

    DEFF Research Database (Denmark)

    Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria;

    2015-01-01

    We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression...

  9. Focus on PTEN regulation

    Directory of Open Access Journals (Sweden)

    Miriam eBermudez-Brito

    2015-07-01

    Full Text Available The role of PTEN as a tumour suppressor has been for a long time attributed to its lipid phosphatase activity against PI(3,4,5P3, the phospholipid product of the class I PI3Ks. Besides its traditional role as a lipid phosphatase at the plasma membrane, a wealth of data has shown that PTEN can function independently of its phosphatase activity and that PTEN also exists and plays a role in the nucleus, in cytoplasmic organelles and extracellularly. Accumulating evidence has shed light on diverse physiological functions of PTEN which are accompanied by a complex regulation of its expression and activity. PTEN levels and function are regulated transcriptionally, post-transcriptionally and post-translationally. PTEN is also sensitive to regulation by its interacting proteins and its localization. Herein, we summarize the current knowledge on mechanisms that regulate the expression and enzymatic activity of PTEN and its role in human diseases.

  10. Post-transcriptional gene silencing of ribosomal protein S6 kinase 1 restores insulin action in leucine-treated skeletal muscle

    DEFF Research Database (Denmark)

    Deshmukh, A; Salehzadeh, F; Metayer-Coustard, S

    2009-01-01

    Excessive nutrients, especially amino acids, impair insulin action on glucose metabolism in skeletal muscle. We tested the hypothesis that the branched-chain amino acid leucine reduces acute insulin action in primary myotubes via a negative feedback mechanism involving ribosomal protein S6 kinase 1...... to excessive leucine. In conclusion, S6K1 plays an important role in the regulation of insulin action on glucose metabolism in skeletal muscle....

  11. Regulation of Nav1.7 : A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia

    NARCIS (Netherlands)

    Koenig, Jennifer; Werdehausen, Robert; Linley, John E; Habib, Abdella M; Vernon, Jeffrey; Lolignier, Stephane; Eijkelkamp, Niels; Zhao, Jing; Okorokov, Andrei L; Woods, C Geoffrey; Wood, John N; Cox, James J

    2015-01-01

    The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conse

  12. B cell differentiation in EBV-positive Burkitt Lymphoma is impaired at post-transcriptional level by miRNA altered expression

    DEFF Research Database (Denmark)

    Leucci, E; Onnis, A; Cocco, M;

    2009-01-01

    Endemic, sporadic and HIV-associated Burkitt lymphoma (BL) all have a B-cell phenotype and a MYC translocation, but a variable association with the Epstein-Barr virus (EBV). However, there is still no satisfactory explanation of how EBV participates in the pathogenesis of BL. A recent investigation...... suggested that EBV-positive and EBV-negative BL have different cells of origin. In particular, according to immunoglobulin gene mutation analysis, EBV-negative BLs may originate from early centroblasts, whereas EBV-positive BLs appear to arise from postgerminal center B cells or memory B cells....... The appearance of a germinal center phenotype in EBV-positive cells might thus derive from a block in B cell differentiation. The exit from the germinal center involves a complex series of events which require the activation of BLIMP-1 and the consequent down-regulation of several target genes.Here, we...

  13. Human T-cell leukemia virus type 2 post-transcriptional control protein p28 is required for viral infectivity and persistence in vivo

    Directory of Open Access Journals (Sweden)

    Kesic Matthew

    2008-05-01

    Full Text Available Abstract Background Human T-cell leukemia virus (HTLV type 1 and type 2 are related but distinct pathogenic complex retroviruses. HTLV-1 is associated with adult T-cell leukemia and a variety of immune-mediated disorders including the chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis. In contrast, HTLV-2 displays distinct biological differences and is much less pathogenic, with only a few reported cases of leukemia and neurological disease associated with infection. In addition to the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex and accessory proteins. Tax and Rex positively regulate virus production and are critical for efficient viral replication and pathogenesis. Using an over-expression system approach, we recently reported that the accessory gene product of the HTLV-1 and HTLV-2 open reading frame (ORF II (p30 and p28, respectively acts as a negative regulator of both Tax and Rex by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and virion production. Further characterization revealed that p28 was distinct from p30 in that it was devoid of major transcriptional modulating activity, suggesting potentially divergent functions that may be responsible for the distinct pathobiologies of HTLV-1 and HTLV-2. Results In this study, we investigated the functional significance of p28 in HTLV-2 infection, proliferation, and immortaliztion of primary T-cells in culture, and viral survival in an infectious rabbit animal model. An HTLV-2 p28 knockout virus (HTLV-2Δp28 was generated and evaluated. Infectivity and immortalization capacity of HTLV-2Δp28 in vitro was indistinguishable from wild type HTLV-2. In contrast, we showed that viral replication was severely attenuated in rabbits inoculated with HTLV-2Δp28 and the mutant virus failed to establish persistent infection. Conclusion We provide direct evidence that p28 is dispensable for

  14. Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor

    Science.gov (United States)

    Chen, Kuan-I.; Pan, Chien-Yuan; Li, Keng-Hui; Huang, Ying-Chih; Lu, Chia-Wei; Tang, Chuan-Yi; Su, Ya-Wen; Tseng, Ling-Wei; Tseng, Kun-Chang; Lin, Chi-Yun; Chen, Chii-Dong; Lin, Shih-Shun; Chen, Yit-Tsong

    2015-11-01

    Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3‧-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

  15. Regulation of cry Gene Expression in Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Chao Deng

    2014-07-01

    Full Text Available Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcriptional, metabolic and post-translational levels.

  16. Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation

    OpenAIRE

    Fields Joshua A; Thompson Stuart A

    2012-01-01

    Abstract Background Although Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previou...

  17. 基因转录后调控在DNA损伤反应中的重要功能%The post-transcriptional regulation of the DNA damage response

    Institute of Scientific and Technical Information of China (English)

    林德玲; 罗瑛; 宋宜

    2014-01-01

    DNA损伤发生时,细胞会激活一系列复杂的信号网络来调控细胞周期检查,完成DNA损伤修复或当损伤超过修复能力时诱导凋亡,这一信号网络被称为DNA损伤反应(DNA damage response,DDR).以往DDR信号网络的研究主要集中于基因转录调控和蛋白共价修饰对功能分子的稳定性和活性调控.近年来,mRNA稳定性调控和mRNA翻译调控等基因转录后调控机制在DDR中的重要作用引起研究者越来越多的关注.研究证明:多种microRNAs和RNA结合蛋白(RNA-binding proteins,RBPs)在转录后水平调控诸多重要功能蛋白的表达,在DDR信号网络中起着不可或缺的作用.文章针对DDR反应中转录后调控的研究进展以及参与其中的microRNAs和RBPs进行阐述和讨论.

  18. The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context

    DEFF Research Database (Denmark)

    Biebinger, S; Rettenmaier, S; Flaspohler, J;

    1996-01-01

    RNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP...... not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid...

  19. Molecular Mechanisms Regulating Macrophage Response to Hypoxia

    Directory of Open Access Journals (Sweden)

    Michal Amit Rahat

    2011-09-01

    Full Text Available Monocytes and Macrophages (Mo/Mϕ exhibit great plasticity, as they can shift between different modes of activation and, driven by their immediate microenvironment, perform divergent functions. These include, among others, patrolling their surroundings and maintaining homeostasis (resident Mo/Mϕ, combating invading pathogens and tumor cells (classically activated or M1 Mo/Mϕ, orchestrating wound healing (alternatively activated or M2 Mo/Mϕ, and restoring homeostasis after an inflammatory response (resolution Mϕ. Hypoxia is an important factor in the Mϕ microenvironment, is prevalent in many physiological and pathological conditions, and is interdependent with the inflammatory response. Although Mo/Mϕ have been studied in hypoxia, the mechanisms by which hypoxia influences the different modes of their activation, and how it regulates the shift between them, remain unclear. Here we review the current knowledge about the molecular mechanisms that mediate this hypoxic regulation of Mϕ activation. Much is known about the hypoxic transcriptional regulatory network, which includes the master regulators HIF-1 and NF-κB, as well as other transcription factors (e.g. AP-1, Erg-1, but we also highlight the role of post-transcriptional and post-translational mechanisms. These mechanisms mediate hypoxic induction of Mϕ pro-angiogenic mediators, suppress M1 Mϕ by post-transcriptionally inhibiting pro-inflammatory mediators, and help shift the classically activated Mϕ into an activation state which approximate the alternatively activated or resolution Mϕ.

  20. K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis.

    Directory of Open Access Journals (Sweden)

    Encarnación Rodríguez-Cazorla

    2015-02-01

    Full Text Available Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK and PEPPER (PEP genes encode RNA-binding proteins that contain three K-homology (KH-domain, the typical configuration of Poly(C-binding ribonucleoproteins (PCBPs. We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC, a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG. Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.

  1. Principles of miRNA-Target Regulation in Metazoan Models

    Directory of Open Access Journals (Sweden)

    Epaminondas Doxakis

    2013-08-01

    Full Text Available MicroRNAs (miRs are key post-transcriptional regulators that silence gene expression by direct base pairing to target sites of RNAs. They have a wide variety of tissue expression patterns and are differentially expressed during development and disease. Their activity and abundance is subject to various levels of control ranging from transcription and biogenesis to miR response elements on RNAs, target cellular levels and miR turnover. This review summarizes and discusses current knowledge on the regulation of miR activity and concludes with novel non-canonical functions that have recently emerged.

  2. Every which way--nanos gene regulation in echinoderms.

    Science.gov (United States)

    Oulhen, Nathalie; Wessel, Gary M

    2014-03-01

    Nanos is an essential factor of germ line success in all animals tested. This gene encodes a Zn-finger RNA-binding protein that in complex with its partner pumilio binds to and changes the fate of several known transcripts. We summarize here the documented functions of Nanos in several key organisms, and then emphasize echinoderms as a working model for how nanos expression is regulated. Nanos presence outside of the target cells is often detrimental to the animal, and in sea urchins, nanos expression appears to be regulated at every step of transcription, and post-transcriptional activity, making this gene product exciting, every which way.

  3. Regulation of L-threonine dehydrogenase in somatic cell reprogramming.

    Science.gov (United States)

    Han, Chuanchun; Gu, Hao; Wang, Jiaxu; Lu, Weiguang; Mei, Yide; Wu, Mian

    2013-05-01

    Increasing evidence suggests that metabolic remodeling plays an important role in the regulation of somatic cell reprogramming. Threonine catabolism mediated by L-threonine dehydrogenase (TDH) has been recognized as a specific metabolic trait of mouse embryonic stem cells. However, it remains unknown whether TDH-mediated threonine catabolism could regulate reprogramming. Here, we report TDH as a novel regulator of somatic cell reprogramming. Knockdown of TDH inhibits, whereas induction of TDH enhances reprogramming efficiency. Moreover, microRNA-9 post-transcriptionally regulates the expression of TDH and thereby inhibits reprogramming efficiency. Furthermore, protein arginine methyltransferase (PRMT5) interacts with TDH and mediates its post-translational arginine methylation. PRMT5 appears to regulate TDH enzyme activity through both methyltransferase-dependent and -independent mechanisms. Functionally, TDH-facilitated reprogramming efficiency is further enhanced by PRMT5. These results suggest that TDH-mediated threonine catabolism controls somatic cell reprogramming and indicate the importance of post-transcriptional and post-translational regulation of TDH.

  4. UCP2, a mitochondrial protein regulated at multiple levels.

    Science.gov (United States)

    Donadelli, Massimo; Dando, Ilaria; Fiorini, Claudia; Palmieri, Marta

    2014-04-01

    An ever-increasing number of studies highlight the role of uncoupling protein 2 (UCP2) in a broad range of physiological and pathological processes. The knowledge of the molecular mechanisms of UCP2 regulation is becoming fundamental in both the comprehension of UCP2-related physiological events and the identification of novel therapeutic strategies based on UCP2 modulation. The study of UCP2 regulation is a fast-moving field. Recently, several research groups have made a great effort to thoroughly understand the various molecular mechanisms at the basis of UCP2 regulation. In this review, we describe novel findings concerning events that can occur in a concerted manner at various levels: Ucp2 gene mutation (single nucleotide polymorphisms), UCP2 mRNA and protein expression (transcriptional, translational, and protein turn-over regulation), UCP2 proton conductance (ligands and post-transcriptional modifications), and nutritional and pharmacological regulation of UCP2.

  5. mTOR regulates the expression of DNA damage response enzymes in long-lived Snell dwarf, GHRKO, and PAPPA-KO mice.

    Science.gov (United States)

    Dominick, Graham; Bowman, Jacqueline; Li, Xinna; Miller, Richard A; Garcia, Gonzalo G

    2017-02-01

    Studies of the mTOR pathway have prompted speculation that diminished mTOR complex-1 (mTORC1) function may be involved in controlling the aging process. Our previous studies have shown diminished mTORC1 activity in tissues of three long-lived mutant mice: Snell dwarf mice, growth hormone receptor gene disrupted mice (GHRKO), and in this article, mice deficient in the pregnancy-associated protein-A (PAPPA-KO). The ways in which lower mTOR signals slow aging and age-related diseases are, however, not well characterized. Here, we show that Snell, GHKRO, and PAPPA-KO mice express high levels of two proteins involved in DNA repair, O-6-methylguanine-DNA methyltransferase (MGMT) and N-myc downstream-regulated gene 1 (NDRG1). Furthermore, we report that lowering mTOR enhances MGMT and NDRG1 protein expression via post-transcriptional mechanisms. We show that the CCR4-NOT complex, a post-transcriptional regulator of gene expression, is downstream of the mTORC1 pathway and may be responsible for the upregulation of MGMT and NDRG1 in all three varieties of long-lived mice. Our data thus suggest a novel link between DNA repair and mTOR signaling via post-transcriptional regulation involving specific alteration in the CCR4-NOT complex, whose modulation could control multiple aspects of the aging process.

  6. UPF2 is a critical regulator of liver development, function and regeneration

    DEFF Research Database (Denmark)

    Thoren, Lina A; Nørgaard, Gitte A; Weischenfeldt, Joachim

    2010-01-01

    BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a post-transcriptional RNA surveillance process that facilitates the recognition and destruction of mRNAs bearing premature terminations codons (PTCs). Such PTC-containing (PTC+) mRNAs may arise from different processes, including erroneous...... processing and expression of pseudogenes, but also from more regulated events such as alternative splicing coupled NMD (AS-NMD). Thus, the NMD pathway serves both as a silencer of genomic noise and a regulator of gene expression. Given the early embryonic lethality in NMD deficient mice, uncovering the full...

  7. Experimental measurements and mathematical modeling of biological noise arising from transcriptional and translational regulation of basic synthetic gene circuits.

    Science.gov (United States)

    Bandiera, Lucia; Pasini, Alice; Pasotti, Lorenzo; Zucca, Susanna; Mazzini, Giuliano; Magni, Paolo; Giordano, Emanuele; Furini, Simone

    2016-04-21

    The small number of molecules, unevenly distributed within an isogenic cell population, makes gene expression a noisy process, and strategies have evolved to deal with this variability in protein concentration and to limit its impact on cellular behaviors. As translational efficiency has a major impact on biological noise, a possible strategy to control noise is to regulate gene expression processes at the post-transcriptional level. In this study, fluctuations in the concentration of a green fluorescent protein were compared, at the single cell level, upon transformation of an isogenic bacterial cell population with synthetic gene circuits implementing either a transcriptional or a post-transcriptional control of gene expression. Experimental measurements showed that protein variability is lower under post-transcriptional control, when the same average protein concentrations are compared. This effect is well reproduced by stochastic simulations, supporting the hypothesis that noise reduction is due to the control mechanism acting on the efficiency of translation. Similar strategies are likely to play a role in noise reduction in natural systems and to be useful for controlling noise in synthetic biology applications.

  8. Synthetic RNAs for gene regulation: design principles and computational tools

    Directory of Open Access Journals (Sweden)

    Alessandro eLaganà

    2014-12-01

    Full Text Available The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies, but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis and the evaluation of RNAi agents such as small-interfering RNA (siRNA, short-hairpin RNA (shRNA, artificial microRNA (a-miR and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats, was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

  9. MicroRNA-30b-mediated regulation of catalase expression in human ARPE-19 cells.

    Directory of Open Access Journals (Sweden)

    Rashidul Haque

    Full Text Available BACKGROUND: Oxidative injury to retinal pigment epithelium (RPE and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD. Reactive oxygen species (ROS-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19 that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H(2O(2 radicals. Exposure to several stress-inducing agents including H(2O(2 has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H(2O(2 (200 µM up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment. CONCLUSIONS/SIGNIFICANCE: We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.

  10. Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB.

    Directory of Open Access Journals (Sweden)

    Audrey Coornaert

    Full Text Available PhoQ/PhoP is a central two-component system involved in magnesium homeostasis, pathogenicity, cell envelope composition, and acid resistance in several bacterial species. The small RNA GcvB is identified here as a novel direct regulator of the synthesis of PhoQ/PhoP in Escherichia coli, and this control relies on a novel pairing region of GcvB. After MicA, this is the second Hfq-dependent small RNA that represses expression of the phoPQ operon. Both MicA and GcvB bind phoPQ mRNA in vivo and in vitro around the translation initiation region of phoP. Binding of either small RNA is sufficient to inhibit ribosome binding and induce mRNA degradation. Surprisingly, however, MicA and GcvB have different effects on the levels of the PhoP protein and therefore on the expression of the PhoP regulon. These results highlight the complex connections between small RNAs and transcriptional regulation networks in bacteria.

  11. MicroRNA regulation of Autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lund, Anders H

    2012-01-01

    recently contributed to our understanding of the molecular mechanisms of the autophagy machinery, yet several gaps remain in our knowledge of this process. The discovery of microRNAs (miRNAs) established a new paradigm of post-transcriptional gene regulation and during the past decade these small non......-coding RNAs have been closely linked to virtually all known fundamental biological pathways. Deregulation of miRNAs can contribute to the development of human diseases, including cancer, where they can function as bona fide oncogenes or tumor suppressors.In this review, we highlight recent advances linking miRNAs...... perspective, but also from a therapeutic view, where miRNAs can be harnessed experimentally to alter autophagy levels in human tumors, affecting parameters such as tumor survival and treatment sensitivity....

  12. Epigenetic microRNA Regulation

    DEFF Research Database (Denmark)

    Wiklund, Erik Digman

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that negatively regulate gene expression post-transcriptionally by binding to complementary sequences in the 3’UTR of target mRNAs in the cytoplasm. However, recent evidence suggests that certain miRNAs are enriched in the nucleus......, and their targets do not seem restricted to mRNA 3’UTRs. Therefore, miRNAs are predicted to have a variety functions throughout mammalian cells. MiRNA genes appear to be regulated in much the same way as coding genes, but current insight into transcriptional miRNA control lacks detail, as mapping miRNA promoters...... and confirming transcriptional start sites can be difficult. Epigenetics, gene regulatory and DNA modification mechanisms not involving a change to the primary sequence, have been implied in the regulation of a number of miRNA loci. Both epigenetic and miRNA signatures are broadly altered in cancer...

  13. microRNA-mediated regulation of the tumor microenvironment

    Science.gov (United States)

    Chou, Jonathan; Shahi, Payam; Werb, Zena

    2013-01-01

    The tumor microenvironment includes cells such as fibroblasts, immune cells, endothelial cells, as well as extracellular matrix (ECM), proteases, and cytokines. Together, these components participate in a complex crosstalk with neoplastic tumor cells that affects growth, angiogenesis, and metastasis. MicroRNAs (miRNAs) are small, non-coding RNAs involved in post-transcriptional regulation of gene expression and have recently emerged as important players involved in regulating multiple aspects of cancer biology and the tumor microenvironment. Differential miRNA expression in both the epithelial and stromal compartments of tumors compared with normal tissue suggests that miRNAs are important drivers of tumorigenesis and metastasis. This review article summarizes our current understanding of the diverse roles of miRNAs involved in tumor microenvironment regulation and underscores the importance of miRNAs within multiple cell types that contribute to the hallmarks of cancer. PMID:24036551

  14. Regulation of biofilm formation in Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Simm, Roger; Ahmad, Irfan; Rhen, Mikael; Le Guyon, Soazig; Römling, Ute

    2014-01-01

    In animals, plants and the environment, Salmonella enterica serovar Typhimurium forms the red dry and rough (rdar) biofilm characterized by extracellular matrix components curli and cellulose. With complex expression control by at least ten transcription factors, the bistably expressed orphan response regulator CsgD directs rdar morphotype development. CsgD expression is an integral part of the Hfq regulon and the complex cyclic diguanosine monophosphate signaling network partially controlled by the global RNA-binding protein CsrA. Cell wall turnover and the periplasmic redox status regulate csgD expression on a post-transcriptional level by unknown mechanisms. Furthermore, phosphorylation of CsgD is a potential inactivation and degradation signal in biofilm dissolution. Including complex incoherent feed-forward loops, regulation of biofilm formation versus motility and virulence is of recognized complexity.

  15. Absence of canonical active chromatin marks in developmentally regulated genes

    Science.gov (United States)

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

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

    Energy Technology Data Exchange (ETDEWEB)

    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 eukaryotic 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?

  17. Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

    Science.gov (United States)

    Alonso, G D; Pereira, C A; Remedi, M S; Paveto, M C; Cochella, L; Ivaldi, M S; Gerez de Burgos, N M; Torres, H N; Flawiá, M M

    2001-06-01

    In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

  18. RNA Splicing: Regulation and Dysregulation in the Heart.

    Science.gov (United States)

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease.

  19. Regulation of spermatogenesis by small non-coding RNAs: role of the germ granule.

    Science.gov (United States)

    de Mateo, Sara; Sassone-Corsi, Paolo

    2014-05-01

    The spermatogenic process relays in highly regulated gene expression mechanisms at the transcriptional and post-transcriptional levels to generate the male gamete that is needed for the perpetuation of the species. Small non-coding RNA pathways have been determined to participate in the post-transcriptional regulatory processes of germ cells. The most important sncRNA molecules that are critically involved in spermatogenesis belong to the miRNA and piRNAs pathways as illustrated by animal models where ablation of specific protein components displays male infertility. Several elements of these regulatory pathways have been found in the nuage or germ granule, a non-membranous cytoplasmatic structure that can be seen in spermatocytes and spermatids. This notion suggests that germ granules may act as organizer centers for silencing pathways in the germline. In general, miRNAs regulate spermatogenesis through targeting and down-regulation of specific transcripts to eventually promote sperm development. However, piRNAs are powerful repressors of transposon elements expression in the spermatogenic process. Here we describe the suggested functions that miRNA and piRNAs pathways execute in the regulation of spermatogenesis and include some recent studies in the field. Despite major strides on the detailed molecular mechanisms of sncRNAs in relation to spermatogenesis, there is plenty to discover on this fascinating regulatory program.

  20. Rbm46 regulates mouse embryonic stem cell differentiation by targeting β-Catenin mRNA for degradation

    Science.gov (United States)

    Zhai, Lei; Wang, Chenchen; Chen, Yuanfan; Zhou, Shixin; Li, Lingsong

    2017-01-01

    Embryonic stem cells (ESCs) are pluripotent cells and have the capability for differentiation into any of the three embryonic germ layers. The Wnt/β-Catenin pathway has been shown to play an essential role in ESC differentiation regulation. Activation of β-Catenin by post-translational modification has been extensively studied. However, mechanism(s) of post-transcriptional regulation of β-Catenin are not well defined. In this study, we report an RNA recognition motif-containing protein (RNA binding motif protein 46, RBM46) which regulates the degradation of β-Catenin mRNA. Our results show that Rbm46 is distributed primarily in the cytoplasm of mouse ESCs (mESCs) and is elevated during the process of ESC differentiation. In addition, overexpression of Rbm46 results in differentiation of mESCs into trophectoderm, while knock-down of Rbm46 leads to mESC differentiation into endoderm. β-Catenin, a key effector in the Wnt pathway which has been reported to play a significant role in the regulation of ESC differentiation, is post-transcriptionally regulated by Rbm46. Our study reveals Rbm46 plays a novel role in the regulation of ESC differentiation. PMID:28212427

  1. Micro-RNA: A New Kind of Gene Regulators

    Institute of Scientific and Technical Information of China (English)

    WU Dan; HU Lan

    2006-01-01

    A group of small RNA molecules, distinct from but related to siRNAs (small interference RNAs) have been identified in a variety of organisms. These small RNAs, called microRNAs (miRNAs), are endogenously encoded approximately 20-24 nt long single-stranded RNAs. They are generally expressed in a highly tissue- or developmental-stage-specific fashion and are post-transcriptional regulator of gene expression in animals and plants. This article summarizes the character, mechanism and analysis method about miRNAs. The current view that miRNAs represent a newly discovered, hidden layer of gene regulation has resulted in high interest among researchers in the discovery of miRNAs, their targets, expression mechanism of action and analysis methods.

  2. Mechanism of T cell regulation by microRNAs

    Institute of Scientific and Technical Information of China (English)

    Juan Liu; Chang-Ping Wu; Bin-Feng Lu; Jing-Ting Jiang

    2013-01-01

    MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that can modulate target gene expression at post-transcriptional level and participate in cell proliferation, differentiation, and apoptosis. T cells have important functions in acquired immune response;miRNAs regulate this immune response by targeting the mRNAs of genes involved in T cell development, proliferation, differentiation, and function. For instance, miR-181 family members function in progression by targeting Bcl2 and CD69, among others. MiR-17 to miR-92 clusters function by binding to CREB1, PTEN, and Bim. Considering that the suppression of T cell-mediated immune responses against tumor cells is involved in cancer progression, we should investigate the mechanism by which miRNA regulates T cells to develop new approaches for cancer treatment.

  3. The Multiple Roles of Microrna-223 in Regulating Bone Metabolism

    Directory of Open Access Journals (Sweden)

    Yong Xie

    2015-10-01

    Full Text Available Bone metabolism is a lifelong process for maintaining skeletal system homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts. Aberrant differentiation of osteoclasts and osteoblasts leads to imbalanced bone metabolism, resulting in ossification and osteolysis diseases. MicroRNAs (miRNAs are pivotal factors in regulating bone metabolism via post-transcriptional inhibition of target genes. Recent studies have revealed that miR-223 exerts multiple effects on bone metabolism, especially in the processes of osteoclast and osteoblasts differentiation. In this review, we highlight the roles of miR-223 during the processes of osteoclast and osteoblast differentiation, as well as the potential clinical applications of miR-223 in bone metabolism disorders.

  4. TDP-43 regulates the microprocessor complex activity during in vitro neuronal differentiation.

    Science.gov (United States)

    Di Carlo, Valerio; Grossi, Elena; Laneve, Pietro; Morlando, Mariangela; Dini Modigliani, Stefano; Ballarino, Monica; Bozzoni, Irene; Caffarelli, Elisa

    2013-12-01

    TDP-43 (TAR DNA-binding protein 43) is an RNA-binding protein implicated in RNA metabolism at several levels. Even if ubiquitously expressed, it is considered as a neuronal activity-responsive factor and a major signature for neurological pathologies, making the comprehension of its activity in the nervous system a very challenging issue. TDP-43 has also been described as an accessory component of the Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) microprocessor complex, which is crucially involved in basal and tissue-specific RNA processing events. In the present study, we exploited in vitro neuronal differentiation systems to investigate the TDP-43 demand for the microprocessor function, focusing on both its canonical microRNA biosynthetic activity and its alternative role as a post-transcriptional regulator of gene expression. Our findings reveal a novel role for TDP-43 as an essential factor that controls the stability of Drosha protein during neuronal differentiation, thus globally affecting the production of microRNAs. We also demonstrate that TDP-43 is required for the Drosha-mediated regulation of Neurogenin 2, a master gene orchestrating neurogenesis, whereas post-transcriptional control of Dgcr8, another Drosha target, resulted to be TDP-43-independent. These results implicate a previously uncovered contribution of TDP-43 in regulating the abundance and the substrate specificity of the microprocessor complex and provide new insights into TDP-43 as a key player in neuronal differentiation.

  5. Regulation of connexins expression levels by microRNAs, an update

    Directory of Open Access Journals (Sweden)

    Juan Francisco Calderon

    2016-11-01

    Full Text Available Control of cell-cell coordination and communication is regulated by several factors, including paracrine and autocrine release of biomolecules, and direct exchange of soluble factors between cells through gap junction channels. Additionally, hemichannels also participate in cell-cell coordination through the release of signaling molecules, such as ATP and glutamate. A family of transmembrane proteins named connexins forms both gap junction channels and hemichannels. Because of their importance in cell and tissue coordination, connexins are controlled both by post-translational and post-transcriptional modifications. In recent years, non-coding RNAs have garnered research interest due to their ability to exert post-transcriptional regulation of gene expression. One of the most recent, well-documented control mechanisms of protein synthesis is found through the action of small, single-stranded RNA, called micro RNAs (miRNAs or miRs. Put simply, miRNAs are negative regulators of the expression of a myriad proteins involved in many physiological and pathological processes. This mini review will briefly summarize what is currently known about the action of miRNAs over Cxs expression/function in different organs under some relevant physiological and pathological conditions

  6. MicroRNAs as regulators in plant metal toxicity response

    Directory of Open Access Journals (Sweden)

    Ana Belen Mendoza-Soto

    2012-05-01

    Full Text Available Metal toxicity is a major stress affecting crop production. This includes metals that are essential for plants (copper, iron, zinc, manganese, and non-essential metals (cadmium, aluminum, cobalt, mercury. A primary common effect of high concentrations of metals such as aluminum, cooper, cadmium or mercury, is root growth inhibition. Metal toxicity triggers the accumulation of reactive oxygen species leading to damage of lipids, proteins and DNA. The plants response to metal toxicity involves several biological processes that require fine and precise regulation at transcriptional and post-transcriptional levels. MicroRNAs (miRNAs are 21 nucleotides non-coding RNAs that regulate gene expression at the post-transcriptional level. A miRNA, incorporated into a RNA induced silencing complex, promotes cleavage of its target mRNA that is recognized by an almost perfect base complementarity. In plants miRNA regulation has been involved in development and also in biotic and abiotic stress responses. We review novel advances in identifying miRNAs related to metal toxicity responses and their potential role according to their targets. Most of the targets for plant metal-responsive miRNAs are transcription factors. Information about metal-responsive miRNAs in different plants points to important regulatory roles of miR319, miR390, miR393 and miR398. The target of miR319 is the TCP transcription factor, implicated in growth control. MiR390 exerts its action through the biogenesis of trans-acting small interference RNAs that, in turn, regulate auxin responsive factors. MiR393 targets the auxin receptors TIR1/AFBs and a bHLH transcription factor. Increasing evidence points to the crucial role of miR398 and its targets Cu/Zn superoxide dismutases in the control of the oxidative stress generated after high metal copper or iron exposure.

  7. Regulation of HMG-CoA reductase in MCF-7 cells by genistein, EPA, and DHA, alone and in combination with mevastatin.

    Science.gov (United States)

    Duncan, Robin E; El-Sohemy, Ahmed; Archer, Michael C

    2005-06-28

    We investigated the regulation of HMG-CoA reductase in MCF-7 human breast cancer cells by genistein, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). All three compounds down-regulated reductase activity, primarily through post-transcriptional effects. In mevastatin-treated cells, only genistein and DHA abrogated the induction of reductase activity caused by this competitive inhibitor. Diets rich in soy isoflavones and fish oils, therefore, may exert anti-cancer effects through the inhibition of mevalonate synthesis in the breast. Genistein and DHA, in particular, may augment the efficacy of statins, increasing the potential for use of these drugs in adjuvant therapy for breast cancer.

  8. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

    OpenAIRE

    Lopez de Silanes, I.; Gorospe, M.; Taniguchi, H; Abdelmohsen, K; Srikantan, S.; Alaminos, M.; Berdasco, M.; Urdinguio, R. G.; Fraga, M. F.; Jacinto, F. V.; Esteller, M.

    2009-01-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3???UTR pr...

  9. Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio

    KAUST Repository

    Gomes-Santos, Carina S. S.

    2011-05-19

    Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism. 2011 Gomes-Santos et al.

  10. Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio.

    Directory of Open Access Journals (Sweden)

    Carina S S Gomes-Santos

    2011-05-01

    Full Text Available Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp., the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2 sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism.

  11. Preventing AID, a physiological mutator, from deleterious activation: regulation of the genomic instability that is associated with antibody diversity.

    Science.gov (United States)

    Nagaoka, Hitoshi; Tran, Thinh Huy; Kobayashi, Maki; Aida, Masatoshi; Honjo, Tasuku

    2010-04-01

    Activation-induced cytidine deaminase (AID) is essential and sufficient to accomplish class-switch recombination and somatic hypermutation, which are two genetic events required for the generation of antibody-mediated memory responses. However, AID can also introduce genomic instability, giving rise to chromosomal translocation and/or mutations in proto-oncogenes. It is therefore important for cells to suppress AID expression unless B lymphocytes are stimulated by pathogens. The mechanisms for avoiding the accidental activation of AID and thereby avoiding genomic instability can be classified into three types: (i) transcriptional regulation, (ii) post-transcriptional regulation and (iii) target specificity. This review summarizes the recently elucidated comprehensive transcriptional regulation mechanisms of the AID gene and the post-transcriptional regulation that may be critical for preventing excess AID activity. Finally, we discuss why AID targets not only Igs but also other proto-oncogenes. AID targets many genes but it is not totally promiscuous and the criteria that specify its targets are unclear. A recent finding that a non-B DNA structure forms upon a decrease in topoisomerase 1 expression may explain this paradoxical target specificity determination. Evolution has chosen AID as a mutator of Ig genes because of its efficient DNA cleavage activity, even though its presence increases the risk of genomic instability. This is probably because immediate protection against pathogens is more critical for species survival than complete protection from the slower acting consequences of genomic instability, such as tumor formation.

  12. MicroRNAs regulate mitochondrial apoptotic pathway in myocardial ischemia-reperfusion-injury.

    Science.gov (United States)

    Makhdoumi, Pouran; Roohbakhsh, Ali; Karimi, Gholamreza

    2016-12-01

    MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene regulators. They are involved in the pathogenesis of different disorders including heart diseases. MiRNAs contribute to ischemia/reperfusion injury (I/RI) by altering numerous key signaling elements. Together with alterations in the various potential signaling pathways, modification in miRNA expression has been suggested as a part of the response network following ischemia/reperfusion (I/R). In addition, cardiac mitochondrial homeostasis is closely associated with cardiac function and impairment of mitochondrial activity occurred after ischemia/reperfusion injury. MiRNAs play a key role in the regulation of mitochondrial apoptotic pathway and signaling proteins. In this review, we summarize the knowledge currently available regarding the molecular mechanisms of miRNA-regulated mitochondrial functions during ischemia/reperfusion injury. This regulation occurs in different stages of mitochondrial apoptosis pathway.

  13. The emerging role of microRNA in regulation of endotoxin tolerance.

    LENUS (Irish Health Repository)

    Quinn, Edel M

    2012-05-01

    Endotoxin tolerance is a phenomenon where cells show reduced responsiveness toward repeated endotoxin stimulation. Regulation of tolerance occurs at multiple levels of the cell signaling cascade, and many of these levels are potentially regulated by miRNA, which are a class of small RNA that bind to mRNA to down-regulate gene expression at the post-transcriptional level. Roles have been identified for miR-146a, miR-221, miR-579, miR-125b, miR-155, let-7e, and miR-98 in regulating the TLR4 signaling pathway during the development of endotoxin tolerance at receptor, signaling pathway, and gene transcription and translational levels. miRNA represent exciting, new potential targets in attempts to exogenously modulate development of endotoxin tolerance.

  14. MicroRNAs regulate osteogenesis and chondrogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Shiwu, E-mail: shiwudong@gmail.com [Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing (China); Yang, Bo; Guo, Hongfeng; Kang, Fei [Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer To focus on the role of miRNAs in chondrogenesis and osteogenesis. Black-Right-Pointing-Pointer Involved in the regulation of miRNAs in osteoarthritis. Black-Right-Pointing-Pointer To speculate some therapeutic targets for bone diseases. -- Abstract: MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potential gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.

  15. Differential Gene Expression and Protein Phosphorylation as Factors Regulating the State of the Arabidopsis SNX1 Protein Complexes in Response to Environmental Stimuli

    Science.gov (United States)

    Brumbarova, Tzvetina; Ivanov, Rumen

    2016-01-01

    Endosomal recycling of plasma membrane proteins contributes significantly to the regulation of cellular transport and signaling processes. Members of the Arabidopsis (Arabidopsis thaliana) SORTING NEXIN (SNX) protein family were shown to mediate the endosomal retrieval of transporter proteins in response to external challenges. Our aim is to understand the possible ways through which external stimuli influence the activity of SNX1 in the root. Several proteins are known to contribute to the function of SNX1 through direct protein–protein interaction. We, therefore, compiled a list of all Arabidopsis proteins known to physically interact with SNX1 and employed available gene expression and proteomic data for a comprehensive analysis of the transcriptional and post-transcriptional regulation of this interactome. The genes encoding SNX1-interaction partners showed distinct expression patterns with some, like FAB1A, being uniformly expressed, while others, like MC9 and BLOS1, were expressed in specific root zones and cell types. Under stress conditions known to induce SNX1-dependent responses, two genes encoding SNX1-interacting proteins, MC9 and NHX6, showed major gene-expression variations. We could also observe zone-specific transcriptional changes of SNX1 under iron deficiency, which are consistent with the described role of the SNX1 protein. This suggests that the composition of potential SNX1-containing protein complexes in roots is cell-specific and may be readjusted in response to external stimuli. On the level of post-transcriptional modifications, we observed stress-dependent changes in the phosphorylation status of SNX1, FAB1A, and CLASP. Interestingly, the phosphorylation events affecting SNX1 interactors occur in a pattern which is largely complementary to transcriptional regulation. Our analysis shows that transcriptional and post-transcriptional regulation play distinct roles in SNX1-mediated endosomal recycling under external stress. PMID:27725825

  16. MicroRNAs: regulators of oncogenesis and stemness

    Directory of Open Access Journals (Sweden)

    Papagiannakopoulos Thales

    2008-06-01

    Full Text Available Abstract MicroRNAs (miRNAs are essential post-transcriptional regulators that determine cell identity and fate. Aberrant expression of miRNAs can lead to diseases, including cancer. Expression of many miRNAs in the de-differentiated brain tumor cancer stem cells resembles that of neural stem cells. In this issue of BMC Medicine, Silber et al provide evidence of the expression of such miRNAs and their potential to mediate differentiation in both stem cell populations. In this commentary, we discuss the known functions of miRNAs in cancer and stem cells, their therapeutic potential and how the findings of Silber et al provide insight into the role of miR-124/miR-137 dysregulation in glioblastomas.

  17. MicroRNAs as regulators of cutaneous wound healing

    Indian Academy of Sciences (India)

    Wing-Fu Lai; Parco M Siu

    2014-06-01

    MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, and have displayed important roles in areas spanning from embryonic development to skin physiology. Despite this, till now little is known about the significance of miRNAs in cutaneous wound healing. In this mini-review, we discuss the existing evidence on the roles of miRNAs in physiological processes relevant to cutaneous wound healing, followed by a highlight of the prospects and challenges of future development of miRNA-based wound therapies. With existing technologies of nucleic acid transfer and miRNA modulation, it is anticipated that once the roles of miRNAs in wound healing have been clarified, there will be a vast new vista of opportunities brought up for development of miRNA-targeted therapies for wound care.

  18. The role of RNases in the regulation of small RNAs.

    Science.gov (United States)

    Saramago, Margarida; Bárria, Cátia; Dos Santos, Ricardo F; Silva, Inês J; Pobre, Vânia; Domingues, Susana; Andrade, José M; Viegas, Sandra C; Arraiano, Cecília M

    2014-04-01

    Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

  19. Plant Nitrogen Acquisition Under Low Availability: Regulation of Uptake and Root Architecture.

    Science.gov (United States)

    Kiba, Takatoshi; Krapp, Anne

    2016-04-01

    Nitrogen availability is a major factor determining plant growth and productivity. Plants acquire nitrogen nutrients from the soil through their roots mostly in the form of ammonium and nitrate. Since these nutrients are scarce in natural soils, plants have evolved adaptive responses to cope with the environment. One of the most important responses is the regulation of nitrogen acquisition efficiency. This review provides an update on the molecular determinants of two major drivers of the nitrogen acquisition efficiency: (i) uptake activity (e.g. high-affinity nitrogen transporters) and (ii) root architecture (e.g. low-nitrogen-availability-specific regulators of primary and lateral root growth). Major emphasis is laid on the regulation of these determinants by nitrogen supply at the transcriptional and post-transcriptional levels, which enables plants to optimize nitrogen acquisition efficiency under low nitrogen availability.

  20. Regulator of myeloid differentiation and function:The secret life of Ikaros

    Institute of Scientific and Technical Information of China (English)

    Olivia; L; Francis; Jonathon; L; Payne; Kimberly; J; Payne

    2011-01-01

    Ikaros (also known as Lyf-1) was initially described as a lymphoid-specific transcription factor.Although Ikaros has been shown to regulate hematopoietic stem cell renewal,as well as the development and function of cells from multiple hematopoietic lineages,including the myeloid lineage,Ikaros has primarily been studied in context of lymphoid development and malignancy.This review focuses on the role of Ikaros in myeloid cells.We address the importance of post-transcriptional regulation of Ikaros function;the emerging role of Ikaros in myeloid malignancy;Ikaros as a regulator of myeloid differentiation and function;and the selective expression of Ikaros isoform-x in cells with myeloid potential.We highlight the challenges of dissecting Ikaros function in lineage commitment decisions among lymphoidmyeloid progenitors that have emerged as a major myeloid differentiation pathway in recent studies,which leads to reconstruction of the traditional map of murine and human hematopoiesis.

  1. Absence of canonical marks of active chromatin in developmentally regulated genes.

    Science.gov (United States)

    Pérez-Lluch, Sílvia; Blanco, Enrique; Tilgner, Hagen; Curado, Joao; Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-10-01

    The interplay of active and repressive histone modifications is assumed to have a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that the transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated with the stable production of RNA, whereas unmarked chromatin would permit rapid gene activation and deactivation during development. In the latter case, regulation by transcription factors would have a comparatively more important regulatory role than chromatin marks.

  2. Post-translational regulation of the β-secretase BACE1.

    Science.gov (United States)

    Araki, Wataru

    2016-09-01

    β-Secretase, widely known as β-site APP cleaving enzyme 1 (BACE1), is a membrane-associated protease that cleaves amyloid precursor protein (APP) to generate amyloid β-protein (Aβ). As this cleavage is a pathologically relevant event in Alzheimer's disease, BACE1 is considered a viable therapeutic target. BACE1 can be regulated at the transcriptional, post-transcriptional, translational, and post-translational levels. Elucidation of the regulatory pathways of BACE1 is critical, not only for understanding the pathological mechanisms of AD but also developing effective therapeutic strategies to inhibit activity of the protease. This mini-review focuses on the post-translational regulation of BACE1, as modulation at this level is closely associated with both physiological and pathological conditions. Current knowledge on the mechanisms underlying such BACE1 regulation and their implications for therapy are discussed.

  3. Inferring RBP-Mediated Regulation in Lung Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Atefeh Lafzi

    Full Text Available RNA-binding proteins (RBPs play key roles in post-transcriptional regulation of mRNAs. Dysregulations in RBP-mediated mechanisms have been found to be associated with many steps of cancer initiation and progression. Despite this, previous studies of gene expression in cancer have ignored the effect of RBPs. To this end, we developed a lasso regression model that predicts gene expression in cancer by incorporating RBP-mediated regulation as well as the effects of other well-studied factors such as copy-number variation, DNA methylation, TFs and miRNAs. As a case study, we applied our model to Lung squamous cell carcinoma (LUSC data as we found that there are several RBPs differentially expressed in LUSC. Including RBP-mediated regulatory effects in addition to the other features significantly increased the Spearman rank correlation between predicted and measured expression of held-out genes. Using a feature selection procedure that accounts for the adaptive search employed by lasso regularization, we identified the candidate regulators in LUSC. Remarkably, several of these candidate regulators are RBPs. Furthermore, majority of the candidate regulators have been previously found to be associated with lung cancer. To investigate the mechanisms that are controlled by these regulators, we predicted their target gene sets based on our model. We validated the target gene sets by comparing against experimentally verified targets. Our results suggest that the future studies of gene expression in cancer must consider the effect of RBP-mediated regulation.

  4. Analysis of cDNAs and Genomic DNA of Snake Venom CTL-like Proteins Revealed An Extraordinary Post-transcriptional Processing Event%蛇毒C型凝集素类蛋白cDNA与基因组DNA序列分析显示特别的转录后加工

    Institute of Scientific and Technical Information of China (English)

    查向东; 周立志; 黄河胜; 刘兢; 徐康森

    2004-01-01

    为研究蛇毒C型凝集素类蛋白的快速进化机制和结构功能关系,使用PCR技术扩增了若干编码C型凝集素类蛋白β链的cDNA分子以及agkisasin β的基因组DNA,并将这些扩增产物进行克隆和测序.对测序结果与试验过程中的具体条件进行了因果关系分析,并且进行点阵图比较和多序列比对.结果表明,可能存在"转录后同源重组"等转录后的事件,在蛇毒C型凝集素类蛋白的多样性上起着重要的作用.对于解释基因数目与蛋白质数目的差异这一后基因组时代的重要问题,具有一定的参考价值.首次报告蛇毒C型凝集素类蛋白的基因组DNA序列,其中未发现有内含子.%To better understand the accelerated evolution of snake venom C-type lectin-like proteins (CTL-like proteins) and to investigate the structure-function relationships, PCR was conducted to amplify cDNAs coding for the β chains of snake venom CTL-like proteins and the genomic DNA of agkisasin β. The reaction products were cloned and sequenced. The causal relationships between the sequences and the experimental conditions were established. Dot plot analysis and multiple alignments were also performed. The results suggested the existence of a post-transcriptional processing event that was essentially homologous recombination at the RNA level, which might play an important role in the diversity of snake venom CTL-like proteins. This inference would provide a novel perspective for explaining a challenging problem of the post-genomic era: the discrepancy between the limited number of genes and the large collection of cDNAs, which was most prominent with regard to certain snake venom proteins. The genomic DNA of a snake venom C-type lectin-like protein was elucidated and no introns were found in the coding region.

  5. MicroRNA, SND1, and alterations in translational regulation in colon carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tsuchiya, Naoto [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Nakagama, Hitoshi, E-mail: hnakagam@ncc.go.jp [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Early Oncogenesis Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2010-11-10

    Post-transcriptional regulation of gene expression by microRNA (miRNA) has recently attracted major interest in relation to its involvement in cancer development. miRNA is a member of small non-coding RNA, consists of 22-24 nucleotides and regulates expression of target mRNA species in a post-transcriptional manner by being incorporated with RNA-induced silencing complex (RISC). Staphylococcal nuclease homology domain containing 1 (SND1), a component of RISC, is frequently up-regulated in human colon cancers and also chemically induced colon cancers in animals. We here showed that SDN1 is involved in miRNA-mediated gene suppression and overexpression of SND1 in colon cancer cells causes down-regulation of APC without altering APC mRNA levels. As for the miRNA expression profile in human colon cancer, miR-34a was among the list of down-regulated miRNA. Expression of miR-34a is tightly regulated by p53, and ectopic expression of miR-34a in colon cancer cells causes remarkable reduction of cell proliferation and induces senescence-like phenotypes. MiR-34a also participates in the positive feedback loop of the p53 tumor suppressor network. This circuitry mechanism for p53 activation is of interest in understanding the tumor suppressive function of miR-34a in colon carcinogenesis. miRNA should also be considered as novel anti-cancer agents in tumor suppressive therapeutic applications.

  6. The Carbon Storage Regulator (Csr) System Exerts a Nutrient-Specific Control over Central Metabolism in Escherichia coli Strain Nissle 1917

    OpenAIRE

    Olga Revelles; Pierre Millard; Jean-Philippe Nougayrède; Ulrich Dobrindt; Eric Oswald; Fabien Létisse; Jean-Charles Portais

    2013-01-01

    The role of the post-transcriptional carbon storage regulator (Csr) system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations) showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the c...

  7. Posttranscriptional regulation of gene expression—adding another layer of complexity to the DNA damage response

    Science.gov (United States)

    Boucas, Jorge; Riabinska, Arina; Jokic, Mladen; Herter-Sprie, Grit S.; Chen, Shuhua; Höpker, Katja; Reinhardt, H. Christian

    2012-01-01

    In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components—a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases. PMID:22936947

  8. Posttranscriptional regulation of gene expression – adding another layer of complexity to the DNA damage response

    Directory of Open Access Journals (Sweden)

    Jorge eBoucas

    2012-08-01

    Full Text Available In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR, is primarily thought to consist of two components – a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RNA-binding proteins and the control of their function through DNA damage-activated protein kinases.

  9. Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle.

    Science.gov (United States)

    Chen, Wenfeng; Liu, Zhenxing; Li, Tianjiao; Zhang, Ruifeng; Xue, Yongbo; Zhong, Yang; Bai, Weiwei; Zhou, Dasen; Zhao, Zhangwu

    2014-11-24

    MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. Here we identify microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of CLOCKWORK ORANGE (CWO). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian prothoracicotropic hormone (PTTH) and CLOCK-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH ecdysteroid receptor. Thus, we find a regulatory cycle involving PTTH, a direct target of CLOCK, and PTTH-driven miRNA let-7.

  10. Patterns of subnet usage reveal distinct scales of regulation in the transcriptional regulatory network of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Carsten Marr

    Full Text Available The set of regulatory interactions between genes, mediated by transcription factors, forms a species' transcriptional regulatory network (TRN. By comparing this network with measured gene expression data, one can identify functional properties of the TRN and gain general insight into transcriptional control. We define the subnet of a node as the subgraph consisting of all nodes topologically downstream of the node, including itself. Using a large set of microarray expression data of the bacterium Escherichia coli, we find that the gene expression in different subnets exhibits a structured pattern in response to environmental changes and genotypic mutation. Subnets with fewer changes in their expression pattern have a higher fraction of feed-forward loop motifs and a lower fraction of small RNA targets within them. Our study implies that the TRN consists of several scales of regulatory organization: (1 subnets with more varying gene expression controlled by both transcription factors and post-transcriptional RNA regulation and (2 subnets with less varying gene expression having more feed-forward loops and less post-transcriptional RNA regulation.

  11. MicroRNA regulation of natural killer cells

    Directory of Open Access Journals (Sweden)

    Ryan eSullivan

    2013-02-01

    Full Text Available Natural Killer (NK cells are innate immune lymphocytes critical for host defense against viral infection and surveillance against malignant transformation. MicroRNAs (miRNAs are a family of small, non-coding RNAs that regulate a wide variety of cellular processes. Recent advances have highlighted the importance of miRNA-mediated post-transcriptional regulation in NK cell development, maturation, and function. This review focuses on several facets of this regulatory mechanism in NK cells: 1 the expressed NK cell miRNA transcriptome; 2 the impact of total miRNA deficiency on NK cells; 3 the role of specific miRNAs regulating NK cell development, survival, and maturation; 4 the intrinsic role of miRNAs regulating NK cell function, including cytokine production, proliferation, and cytotoxicity; and 5 the role of NK cell miRNAs in disease. Currently our knowledge of how miRNAs regulate NK cell biology is limited, and thus we also explore key open questions in the field, as well as approaches and techniques to ascertain the role of individual miRNAs as important molecular regulators.

  12. MicroRNA regulation of F-box proteins and its role in cancer.

    Science.gov (United States)

    Wu, Zhao-Hui; Pfeffer, Lawrence M

    2016-02-01

    MicroRNAs (miRNAs) are small endogenous non-coding RNAs, which play critical roles in cancer development by suppressing gene expression at the post-transcriptional level. In general, oncogenic miRNAs are upregulated in cancer, while miRNAs that act as tumor suppressors are downregulated, leading to decreased expression of tumor suppressors and upregulated oncogene expression, respectively. F-box proteins function as the substrate-recognition components of the SKP1-CUL1-F-box (SCF)-ubiquitin ligase complex for the degradation of their protein targets by the ubiquitin-proteasome system. Therefore F-box proteins and miRNAs both negatively regulate target gene expression post-transcriptionally. Since each miRNA is capable of fine-tuning the expression of multiple target genes, multiple F-box proteins may be suppressed by the same miRNA. Meanwhile, one F-box proteins could be regulated by several miRNAs in different cancer types. In this review, we will focus on miRNA-mediated downregulation of various F-box proteins, the resulting stabilization of F-box protein substrates and the impact of these processes on human malignancies. We provide insight into how the miRNA: F-box protein axis may regulate cancer progression and metastasis. We also consider the broader role of F-box proteins in the regulation of pathways that are independent of the ubiquitin ligase complex and how that impacts on oncogenesis. The area of miRNAs and the F-box proteins that they regulate in cancer is an emerging field and will inform new strategies in cancer treatment.

  13. Translational regulation of Yersinia enterocolitica mRNA encoding a type III secretion substrate.

    Science.gov (United States)

    Kopaskie, Karyl S; Ligtenberg, Katherine Given; Schneewind, Olaf

    2013-12-06

    Yersinia enterocolitica type III secretion machines transport YopQ and other Yop effectors into host immune cells. YopD and its chaperone LcrH are essential components of the Yersinia type III pathway, enabling effector translocation into host cells. YopD, LcrH, and YscM1 also regulate yop expression post-transcriptionally in response to environmental signals; however, the molecular mechanisms for this regulation and Yop secretion are unknown. We show here that YopD associates with 30 S ribosomal particles in a manner requiring LcrH. When added to ribosomes, YopD, LcrH, and YscM1 block the translation of yopQ mRNA. We propose a model whereby LcrH-dependent association of YopD with 30 S ribosomal particles enables YscM1 to block yopQ translation unless type III machines are induced to secrete the effector.

  14. Regulation of gene expression in neuronal tissue by RNA interference and editing

    DEFF Research Database (Denmark)

    Venø, Morten Trillingsgaard

    No tissue in the mammalian organism is more complex than the brain. This complexity is in part the result of precise timing and interplay of a large number mechanisms modulating gene expression post-transcriptionally. Fine-tuning mechanisms such as A-to-I editing of RNA transcripts and regulation...... mediated by microRNAs are crucial for the correct function of the mammalian brain. We are addressing A-to-I editing and regulation by microRNAs with spatio-temporal resolution in the embryonic porcine brain by Solexa sequencing of microRNAs and 454 sequencing of edited neuronal messenger RNAs, resulting...... in detailed data of both of these fine-tuning mechanisms in the embryonic development of the pig. Editing levels of transcripts examined are generally seen to increase through development, in agreement with editing of specific microRNA also examined in the Solexa sequencing study. Three studies examining...

  15. Poly(ADP-ribose) polymerase-13 and RNA regulation in immunity and cancer.

    Science.gov (United States)

    Todorova, Tanya; Bock, Florian J; Chang, Paul

    2015-06-01

    Post-transcriptional regulation of RNA is an important mechanism for activating and resolving cellular stress responses. Poly(ADP-ribose) polymerase-13 (PARP13), also known as ZC3HAV1 and zinc-finger antiviral protein (ZAP), is an RNA-binding protein that regulates the stability and translation of specific mRNAs, and modulates the miRNA silencing pathway to globally affect miRNA targets. These functions of PARP13 are important components of the cellular response to stress. In addition, the ability of PARP13 to restrict oncogenic viruses and to repress the prosurvival cytokine receptor tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 4 (TRAILR4) suggests that it can be protective against malignant transformation and cancer development. The relevance of PARP13 to human health and disease make it a promising therapeutic target.

  16. The cell cycle regulated transcriptome of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Stuart K Archer

    Full Text Available Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a "double-cut" elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.

  17. 植物MEP途径的代谢调控机制%Multiple Regulation Mechanisms of MEP Pathway in Plant

    Institute of Scientific and Technical Information of China (English)

    张松涛; 陈红丽; 崔红; 杨惠娟; 刘国顺

    2012-01-01

    Terpenoids metabolism is one of the most important pathways of secondary metabolism in plant. The regulatory mechanisms that modulate this metabolic route will determine plant growth and development, resistance, quality and other aspects. The terpene precursors are synthesized by the 2-C-Methyl-D-E-rythritol-4-Phosphate (MEP) pathway in plant plastids. Recent studies have shown that, many genes involved in MEP pathway are not only regulated by multiple genes encoding and the transcript level,but also by post-transcriptional mechanism. Post-transcriptional regulation is a novel regulation mechanism described recently for this way and the mechanism is not clear. We review here various regulatory mechanisms of MEP pathway in plant, especially the mechanism and signal molecular that may be involved in post-transcriptional regulation,which may provide the theory basis for the research of regulation in this pathway.%萜类代谢途径是植物中最重要的次生代谢途径之一,对其有效的调控决定着植物的生长发育、抗性及品质等各个方面.植物中类萜合成的前体物在质体中是由2-C-甲基-D-赤藓糖醇-4-磷酸(2-C-Methyl-D-Erythritol-4-Phosphate,MEP)途径合成的,MEP途径中的许多基因除了受到多基因编码和转录水平的调节外,还受到转录后调节机制的调节,而转录后调节是一种新发现的调节方式,其机制还不是很清楚.该文重点对近年来国内外有关植物MEP途径的多种调节方式,尤其是转录后调节的调节机制及其可能参与的信号分子方面的研究进展进行综述,为植物的MEP途径的代谢调控提供参考.

  18. The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Rui Xiang

    2012-02-01

    Full Text Available MicroRNAs (miRNAs have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92 cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2 is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3’ untranslated regions (3’UTR of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3’UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3’UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.

  19. Epigenetic regulation in alcoholic liver disease

    Institute of Scientific and Technical Information of China (English)

    Pranoti Mandrekar

    2011-01-01

    Alcoholic liver disease (ALD) is characterized by steatosis or fat deposition in the liver and inflammation, which leads to cirrhosis and hepatocellular carcinoma. Induction of target genes without involving changes in DNA sequence seems to contribute greatly to liver injury. Chromatin modifications including alterations in histones and DNA, as well as post-transcriptional changes collectively referred to as epigenetic effects are altered by alcohol. Recent studies have pointed to a significant role for epigenetic mechanisms at the nucleosomal level influencing gene expression and disease outcome in ALD. Specifically, epigenetic alterations by alcohol include histone modifications such as changes in acetylation and phosphorylation, hypomethylation of DNA, and alterations in miRNAs. These modifications can be induced by alcohol-induced oxidative stress that results in altered recruitment of transcriptional machinery and abnormal gene expression. Delineating these mechanisms in initiation and progression of ALD is becoming a major area of interest. This review summarizes key epigenetic mechanisms that are dysregulated by alcohol in the liver. Alterations by alcohol in histone and DNA modifications, enzymes related to histone acetylation such as histone acetyltransferases, histone deacetylases and sirtuins, and methylation enzymes such as DNA methyltransferases are discussed. Chromatin modifications and miRNA alterations that result in immune cell dysfunction contributing to inflammatory cytokine production in ALD is reviewed. Finally, the role of alcohol-mediated oxidative stress in epigenetic regulation in ALD is described. A better understanding of these mechanisms is crucial for designing novel epigenetic based therapies to ameliorate ALD.

  20. Hepatic drug transporters and nuclear receptors: Regulation by therapeutic agents

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The canalicular membrane represents the excretory pole of hepatocytes. Bile is an important route of elimina-tion of potentially toxic endo- and xenobiotics (including drugs and toxins), mediated by the major canalicular transporters: multidrug resistance protein 1 (MDR1, ABCB1), also known as P-glycoprotein, multidrug re-sistance-associated protein 2 (MRP2, ABCC2), and the breast cancer resistance protein (BCRP, ABCG2). Their activities depend on regulation of expression and proper localization at the canalicular membrane, as regulated by transcriptional and post-transcriptional events, re-spectively. At transcriptional level, specific nuclear re-ceptors (NR)s modulated by ligands, co-activators and co-repressors, mediate the physiological requirements of these transporters. This complex system is also re-sponsible for alterations occurring in specific liver pa-thologies. We briefly describe the major Class Ⅱ NRs, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), and their role in regulating expression of multidrug resistance proteins. Several therapeutic agents regulate the expression of relevant drug trans-porters through activation/inactivation of these NRs. We provide some representative examples of the action of therapeutic agents modulating liver drug transporters, which in addition, involve CAR or PXR as mediators.

  1. Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

    Directory of Open Access Journals (Sweden)

    Barbara Mojsa

    2014-05-01

    Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

  2. Regulation of Salmonella enterica pathogenicity island 1 by DNA adenine methylation.

    Science.gov (United States)

    López-Garrido, Javier; Casadesús, Josep

    2010-03-01

    DNA adenine methylase (Dam(-)) mutants of Salmonella enterica are attenuated in the mouse model and present multiple virulence-related defects. Impaired interaction of Salmonella Dam(-) mutants with the intestinal epithelium has been tentatively correlated with reduced secretion of pathogenicity island 1 (SPI-1) effectors. In this study, we show that S. enterica Dam(-) mutants contain lowered levels of the SPI-1 transcriptional regulators HilA, HilC, HilD, and InvF. Epistasis analysis indicates that Dam-dependent regulation of SPI-1 requires HilD, while HilA, HilC, and InvF are dispensable. A transcriptional hilDlac fusion is expressed at similar levels in Dam(+) and Dam(-) hosts. However, lower levels of hilD mRNA are found in a Dam(-) background, thus providing unsuspected evidence that Dam methylation might exert post-transcriptional regulation of hilD expression. This hypothesis is supported by the following lines of evidence: (i) lowered levels of hilD mRNA are found in Salmonella Dam(-) mutants when hilD is transcribed from a heterologous promoter; (ii) increased hilD mRNA turnover is observed in Dam(-) mutants; (iii) lack of the Hfq RNA chaperone enhances hilD mRNA instability in Dam(-) mutants; and (iv) lack of the RNA degradosome components polynucleotide phosphorylase and ribonuclease E suppresses hilD mRNA instability in a Dam(-) background. Our report of Dam-dependent control of hilD mRNA stability suggests that DNA adenine methylation plays hitherto unknown roles in post-transcriptional control of gene expression.

  3. The Cln3 cyclin is down-regulated by translational repression and degradation during the G1 arrest caused by nitrogen deprivation in budding yeast.

    Science.gov (United States)

    Gallego, C; Garí, E; Colomina, N; Herrero, E; Aldea, M

    1997-12-01

    Nutrients are among the most important trophic factors in all organisms. When deprived of essential nutrients, yeast cells use accumulated reserves to complete the current cycle and arrest in the following G1 phase. We show here that the Cln3 cyclin, which has a key role in the timely activation of SBF (Swi4-Swi6)- and MBF (Mbp1-Swi6)-dependent promoters in late G1, is down-regulated rapidly at a post-transcriptional level in cells deprived of the nitrogen source. In addition to the fact that Cln3 is degraded faster by ubiquitin-dependent mechanisms, we have found that translation of the CLN3 mRNA is repressed approximately 8-fold under nitrogen deprivation conditions. As a consequence, both SBF- and MBF-dependent expression is strongly down-regulated. Mainly because of their transcriptional dependence on SBF, and perhaps with the contribution of similar post-transcriptional mechanisms to those found for Cln3, the G1 cyclins Cln1 and 2 become undetectable in starved cells. The complete loss of Cln cyclins and the sustained presence of the Clb-cyclin kinase inhibitor Sic1 in starved cells may provide the molecular basis for the G1 arrest caused by nitrogen deprivation.

  4. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

    Directory of Open Access Journals (Sweden)

    Pegine Walrad

    2009-02-01

    Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

  5. Complex regulation of autophagy in cancer - integrated approaches to discover the networks that hold a double-edged sword.

    Science.gov (United States)

    Kubisch, János; Türei, Dénes; Földvári-Nagy, László; Dunai, Zsuzsanna A; Zsákai, Lilian; Varga, Máté; Vellai, Tibor; Csermely, Péter; Korcsmáros, Tamás

    2013-08-01

    Autophagy, a highly regulated self-degradation process of eukaryotic cells, is a context-dependent tumor-suppressing mechanism that can also promote tumor cell survival upon stress and treatment resistance. Because of this ambiguity, autophagy is considered as a double-edged sword in oncology, making anti-cancer therapeutic approaches highly challenging. In this review, we present how systems-level knowledge on autophagy regulation can help to develop new strategies and efficiently select novel anti-cancer drug targets. We focus on the protein interactors and transcriptional/post-transcriptional regulators of autophagy as the protein and regulatory networks significantly influence the activity of core autophagy proteins during tumor progression. We list several network resources to identify interactors and regulators of autophagy proteins. As in silico analysis of such networks often necessitates experimental validation, we briefly summarize tractable model organisms to examine the role of autophagy in cancer. We also discuss fluorescence techniques for high-throughput monitoring of autophagy in humans. Finally, the challenges of pharmacological modulation of autophagy are reviewed. We suggest network-based concepts to overcome these difficulties. We point out that a context-dependent modulation of autophagy would be favored in anti-cancer therapy, where autophagy is stimulated in normal cells, while inhibited only in stressed cancer cells. To achieve this goal, we introduce the concept of regulo-network drugs targeting specific transcription factors or miRNA families identified with network analysis. The effect of regulo-network drugs propagates indirectly through transcriptional or post-transcriptional regulation of autophagy proteins, and, as a multi-directional intervention tool, they can both activate and inhibit specific proteins in the same time. The future identification and validation of such regulo-network drug targets may serve as novel intervention

  6. ATF2, a paradigm of the multifaceted regulation of transcription factors in biology and disease.

    Science.gov (United States)

    Watson, Gregory; Ronai, Ze'ev; Lau, Eric

    2017-02-15

    Stringent transcriptional regulation is crucial for normal cellular biology and organismal development. Perturbations in the proper regulation of transcription factors can result in numerous pathologies, including cancer. Thus, understanding how transcription factors are regulated and how they are dysregulated in disease states is key to the therapeutic targeting of these factors and/or the pathways that they regulate. Activating transcription factor 2 (ATF2) has been studied in a number of developmental and pathological conditions. Recent findings have shed light on the transcriptional, post-transcriptional, and post-translational regulatory mechanisms that influence ATF2 function, and thus, the transcriptional programs coordinated by ATF2. Given our current knowledge of its multiple levels of regulation and function, ATF2 represents a paradigm for the mechanistic complexity that can regulate transcription factor function. Thus, increasing our understanding of the regulation and function of ATF2 will provide insights into fundamental regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into a genomic response through transcription factors. Characterization of ATF2 dysfunction in the context of pathological conditions, particularly in cancer biology and response to therapy, will be important in understanding how pathways controlled by ATF2 or other transcription factors might be therapeutically exploited. In this review, we provide an overview of the currently known upstream regulators and downstream targets of ATF2.

  7. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F;

    2001-01-01

    The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma...... and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested...... of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m...

  8. Single-Cell and Single-Molecule Analysis of Gene Expression Regulation

    Science.gov (United States)

    Vera, Maria; Biswas, Jeetayu; Senecal, Adrien

    2016-01-01

    Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology. PMID:27893965

  9. MicroRNAs: not ‘fine-tuners’ but key regulators of neuronal development and function

    Directory of Open Access Journals (Sweden)

    Gregory eDavis

    2015-11-01

    Full Text Available microRNAs (miRNAs are a class of short non-coding RNAs that operate as prominent post-transcriptional regulators of eukaryotic gene expression. miRNAs are abundantly expressed in the brain of most animals and exert diverse roles. The anatomical and functional complexity of brain requires the precise coordination of multi-layered gene regulatory networks. The flexibility, speed and reversibility of miRNA function provide precise temporal and spatial gene regulatory capabilities that are crucial for the correct functioning of the brain. Studies have shown that the underlying molecular mechanisms controlled by miRNAs in the nervous systems of invertebrate and vertebrate models are remarkably conserved in humans. We endeavour to provide insight into the roles of miRNAs in the nervous systems of these model organisms and discuss how such information may be used to inform regarding diseases of the human brain.

  10. Long non-coding RNAs, a new important regulator of cardiovascular physiology and pathology.

    Science.gov (United States)

    Ma, Yidi; Ma, Wenya; Huang, Lina; Feng, Dan; Cai, Benzhi

    2015-06-01

    LncRNAs were previously considered to be the 'noise' of gene transcription having no biological functions, but now it has become evident that lncRNAs function as modulators of gene expression network. LncRNAs may regulate diverse gene expression levels which were roughly summarized to epigenetic, transcriptional and post-transcriptional levels. It has been clarified that some lncRNAs were expressed differentially in cardiovascular diseases, and aberrant changes of those lncRNAs were involved in the development of heart disorders. The role of lncRNAs in this process transcended the tradition of protein regulatory platform to be the orchestrator of cardiac sophisticated governing system of heart development, adaptation and pathological reaction. This review summarizes recent advances in the study of functions and mechanisms of lncRNAs in cardiovascular physiology and pathology. The regulatory roles of lncRNAs in cardiovascular diseases provide new strategy for interventional therapy of heart diseases.

  11. Molecular Mechanisms Regulating Hepcidin Revealed by Hepcidin Disorders

    Directory of Open Access Journals (Sweden)

    Clara Camaschella

    2011-01-01

    Full Text Available Iron is essential for human life, but toxic if present in excess. To avoid iron overload and maintain iron homeostasis, all cells are able to regulate their iron content through the post-transcriptional control of iron genes operated by the cytosolic iron regulatory proteins that interact with iron responsive elements on iron gene mRNA. At the systemic level, iron homeostasis is regulated by the liver peptide hepcidin. Disruption of these regulatory loops leads to genetic diseases characterized by iron deficiency (iron-refractory iron-deficiency anemia or iron overload (hemochromatosis. Alterations of the same systems are also found in acquired disorders, such as iron-loading anemias characterized by ineffective erythropoiesis and anemia of chronic diseases (ACD associated with common inflammatory conditions. In ACD, iron is present in the body, but maldistributed, being deficient for erythropoiesis, but sequestered in macrophages. Studies of the hepcidin regulation by iron and inflammatory cytokines are revealing new pathways that might become targets of new therapeutic intervention in iron disorders.

  12. The regulation of matrix metalloproteinases and their inhibitors.

    Science.gov (United States)

    Clark, Ian M; Swingler, Tracey E; Sampieri, Clara L; Edwards, Dylan R

    2008-01-01

    The matrix metalloproteinases (MMP) are a family of 23 enzymes in man. These enzymes were originally described as cleaving extracellular matrix (ECM) substrates with a predominant role in ECM homeostasis, but it is now clear that they have much wider functionality. Control over MMP and/or tissue inhibitor of metalloproteinases (TIMP) activity in vivo occurs at different levels and involves factors such as regulation of gene expression, activation of zymogens and inhibition of active enzymes by specific inhibitors. Whilst these enzymes and inhibitors have clear roles in physiological tissue turnover and homeostasis, if control of their expression or activity is lost, they contribute to a number of pathologies including e.g. cancer, arthritis and cardiovascular disease. The expression of many MMPs and TIMPs is regulated at the level of transcription by a variety of growth factors, cytokines and chemokines, though post-transcriptional pathways may contribute to this regulation in specific cases. The contribution of epigenetic modifications has also been uncovered in recent years. The promoter regions of many of these genes have been, at least partly, characterised including the role of identified single nucleotide polymorphisms. This article aims to review current knowledge across these gene families and use a bioinformatic approach to fill the gaps where no functional data are available.

  13. The role of miR-182 in regulating pineal CLOCK expression after hypoxia-ischemia brain injury in neonatal rats.

    Science.gov (United States)

    Ding, Xin; Sun, Bin; Huang, Jian; Xu, Lixiao; Pan, Jian; Fang, Chen; Tao, Yanfang; Hu, Shukun; Li, Ronghu; Han, Xing; Miao, Po; Wang, Ying; Yu, Jian; Feng, Xing

    2015-03-30

    Circadian rhythm disorder is a common neurological deficit caused by neonatal hypoxic-ischemic brain damage (HIBD). However, little is known about its underlying mechanisms. Our previous studies revealed a significant elevation of clock genes at the protein, but not mRNA, levels in the pineal gland after neonatal HIBD. To investigate the mechanisms of post-transcriptional regulation on clock genes, we screened changes of miRNA levels in the pineal gland after neonatal HIBD using high-throughput arrays. Within the miRNAs whose expression was significantly down-regulated, we identified one miRNA (miR182) that targeted the 3'-untranslated region (3'-UTR) of Clock, a key component of clock genes, and played a crucial role in regulating CLOCK expression after oxygen-glucose deprivation in primarily cultured pinealocytes. Our findings therefore provide new insight on studies of therapeutic targets for circadian rhythm disturbance after neonatal HIBD.

  14. Identification of miR-148a as a novel regulator of cholesterol metabolism

    Science.gov (United States)

    Goedeke, Leigh; Rotllan, Noemi; Canfrán-Duque, Alberto; Aranda, Juan F.; Ramírez, Cristina M.; Araldi, Elisa; Lin, Chin-Sheng; Anderson, Norma N.; Wagschal, Alexandre; de Cabo, Rafael; Horton, Jay D.; Lasunción, Miguel A.; Näär, Anders M.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-01-01

    The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL-cholesterol (LDL-C). While the transcriptional regulation of LDLR is well-characterized, the post-transcriptional mechanisms which govern LDLR expression are just beginning to emerge. Here, we developed a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen, we characterize miR-148a as a negative regulator of LDLR expression and activity, and define a novel SREBP1-mediated pathway by which miR-148a regulates LDL-C uptake. Importantly, inhibition of miR-148a increases hepatic LDLR expression and decreases plasma LDL-C in vivo. We also provide evidence that miR-148a regulates hepatic ABCA1 expression and circulating HDL-C levels. Collectively, these studies uncover miR-148a as an important regulator of hepatic LDL-C clearance through direct regulation of LDLR expression, and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate the elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease. PMID:26437365

  15. Dynamic light regulation of translation status in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Julia eBailey-Serres

    2012-04-01

    Full Text Available Light, a dynamic environmental parameter, is an essential regulator of plant growth and development. Light-regulated transcriptional networks are well documented, whereas light-regulated post-transcriptional regulation has received only limited attention. In this study, dynamics in translation of cytosolic mRNAs were evaluated at the genome-level in Arabidopsis thaliana seedlings grown under a typical light / dark diurnal regime, shifted to darkness at midday and then re-illuminated. One-hour of unanticipated darkness reduced levels of polyribosomes (polysomes by 17% in a manner consistent with inhibition of initiation of translation. This down-regulation of protein synthesis was reversed within 10 minutes of re-illumination. Quantitative comparison of the total cellular population of transcripts (the transcriptome to those associated with one or more 80S ribosome (the translatome identified over 1600 mRNAs that are differentially translated in response to light availability. Unanticipated darkness limited transcription and translation of mRNAs encoding components of the photosynthetic machinery. Many mRNAs encoding proteins associated with the energy demanding process of protein synthesis were stable but sequestered in the dark, in a rapidly reversible manner. A meta-analysis determined these same transcripts were similarly and coordinately regulated in response to changes in oxygen availability. The dark and hypoxia translationally repressed mRNAs lack highly supported candidate RNA-regulatory elements but are characterized by G+C-rich 5’-untranslated regions. We propose that dynamic regulation of the translational status of a subset of cellular mRNAs serves as a general energy conservation mechanism.

  16. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    Science.gov (United States)

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  17. Regulation of the mRNA-binding protein HuR by posttranslational modification: spotlight on phosphorylation.

    Science.gov (United States)

    Eberhardt, Wolfgang; Doller, Anke; Pfeilschifter, Josef

    2012-06-01

    The ubiquitous mRNA-binding protein human antigen R (HuR) and its neuronal relatives (HuB, HuC, HuD) participate in the post-transcriptional regulation of many AU-rich element-bearing mRNAs. In addition to its originally described role in controlling mRNA decay, the binding of HuR to target mRNAs can affect many aspects of mRNA processing including splicing, polyadenylation, intracellular trafficking, translation and modulation of mRNA repression by miRNAs. In accordance to the growing list of signalling events which are involved in regulating these different HuR functions, recent data implicate that posttranslational modification, namely protein kinase-triggered phosphorylation of HuR plays a crucial role in connecting extracellular signal inputs to a specific post-transcriptional program by HuR. Notably, in addition to directly targeting HuR functions, posttranslational modifications of HuR have a major impact on the sequestration and binding to various HuR ligand proteins as has been demonstrated e.g. for the 14-3-3 chaperones. However, the detailed mechanisms of how a specific modification of HuR coordinates different aspects in HuR regulation are currently poorly understood. Due to the fact that most of the described HuR activities are closely related to its subcellular localization and the binding to cargo mRNA, this review will focus on these aspects of HuR functions and their control by posttranslational modification, particularly by HuR phosphorylations by different protein kinases.

  18. MicroRNAs: Key Regulators in the Central Nervous System and Their Implication in Neurological Diseases

    Directory of Open Access Journals (Sweden)

    Dan-Dan Cao

    2016-05-01

    Full Text Available MicroRNAs (miRNAs are a class of small, well-conserved noncoding RNAs that regulate gene expression post-transcriptionally. They have been demonstrated to regulate a lot of biological pathways and cellular functions. Many miRNAs are dynamically regulated during central nervous system (CNS development and are spatially expressed in adult brain indicating their essential roles in neural development and function. In addition, accumulating evidence strongly suggests that dysfunction of miRNAs contributes to neurological diseases. These observations, together with their gene regulation property, implicated miRNAs to be the key regulators in the complex genetic network of the CNS. In this review, we first focus on the ways through which miRNAs exert the regulatory function and how miRNAs are regulated in the CNS. We then summarize recent findings that highlight the versatile roles of miRNAs in normal CNS physiology and their association with several types of neurological diseases. Subsequently we discuss the limitations of miRNAs research based on current studies as well as the potential therapeutic applications and challenges of miRNAs in neurological disorders. We endeavor to provide an updated description of the regulatory roles of miRNAs in normal CNS functions and pathogenesis of neurological diseases.

  19. MicroRNAs: Key Regulators in the Central Nervous System and Their Implication in Neurological Diseases.

    Science.gov (United States)

    Cao, Dan-Dan; Li, Lu; Chan, Wai-Yee

    2016-05-28

    MicroRNAs (miRNAs) are a class of small, well-conserved noncoding RNAs that regulate gene expression post-transcriptionally. They have been demonstrated to regulate a lot of biological pathways and cellular functions. Many miRNAs are dynamically regulated during central nervous system (CNS) development and are spatially expressed in adult brain indicating their essential roles in neural development and function. In addition, accumulating evidence strongly suggests that dysfunction of miRNAs contributes to neurological diseases. These observations, together with their gene regulation property, implicated miRNAs to be the key regulators in the complex genetic network of the CNS. In this review, we first focus on the ways through which miRNAs exert the regulatory function and how miRNAs are regulated in the CNS. We then summarize recent findings that highlight the versatile roles of miRNAs in normal CNS physiology and their association with several types of neurological diseases. Subsequently we discuss the limitations of miRNAs research based on current studies as well as the potential therapeutic applications and challenges of miRNAs in neurological disorders. We endeavor to provide an updated description of the regulatory roles of miRNAs in normal CNS functions and pathogenesis of neurological diseases.

  20. SUMO: regulating the regulator

    Directory of Open Access Journals (Sweden)

    Bossis Guillaume

    2006-06-01

    Full Text Available Abstract Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii global regulation of SUMO conjugation and deconjugation.

  1. Posttranscriptional down-regulation of small ribosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency.

    Science.gov (United States)

    Badhai, Jitendra; Fröjmark, Anne-Sophie; Razzaghian, Hamid Reza; Davey, Edward; Schuster, Jens; Dahl, Niklas

    2009-06-18

    Ribosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that small interfering RNA (siRNA) knock-down of RPS19 results in a relative decrease of small subunit (SSU) r-proteins (S20, S21 and S24) when compared to large subunit (LSU) r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post transcriptional regulation of r-proteins at the level of subunit formation.

  2. Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis

    Directory of Open Access Journals (Sweden)

    J. Travis Hinson

    2016-12-01

    Full Text Available AMP-activated protein kinase (AMPK is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling.

  3. miR-181b as a key regulator of the oncogenic process and its clinical implications in cancer (Review).

    Science.gov (United States)

    Liu, Juan; Shi, Weifeng; Wu, Changping; Ju, Jingfang; Jiang, Jingting

    2014-01-01

    MicroRNAs (miRNAs or miRs) are small, non-coding, single-stranded RNA molecules that regulate gene expression at the post-transcriptional level to repress protein expression of target genes. Among these, miR-181b has been found to be a critical regulatory miRNA linking inflammation and cancer. The functional significance of miR-181b in various tumors and translational research suggests that it exhibits great potential as a predictive and prognostic biomarker. Extensive efforts are underway to identify mRNA targets and the affected regulatory networks, which may be the key to providing a better understanding of miR-181b-mediated signaling pathways.

  4. MicroRNA-450a-3p represses cell proliferation and regulates embryo development by regulating Bub1 expression in mouse.

    Directory of Open Access Journals (Sweden)

    Min Luo

    Full Text Available Bub1 is a critical component of the spindle assembly checkpoint (SAC and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3'-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.

  5. Two host microRNAs influence WSSV replication via STAT gene regulation

    Science.gov (United States)

    Huang, Ying; Wang, Wen; Ren, Qian

    2016-01-01

    MicroRNAs (miRNAs) have important roles in post-transcriptional regulation of gene expression. During viral infection, viruses utilize hosts to enhance their replication by altering cellular miRNAs. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays crucial roles in the antiviral responses. In this study, two miRNAs (miR-9041 and miR-9850) from Macrobrachium rosenbergii were found to promote white spot syndrome virus (WSSV) replication. The up-regulation of miR-9041 or miR-9850 suppresses STAT expression in the gills of M. rosenbergii, which subsequently down-regulates the expression of its downstream dynamin (Dnm) genes: Dnm1, Dnm2, and Dnm3. Knockdown of miR-9041 and miR-9850 restricts WSSV replication by up-regulating STAT and Dnm gene expression. The silencing of STAT, Dnm1, Dnm2, or Dnm3 led to an increase of the number of WSSV copies in shrimp. The injection of recombinant Dnm1, Dnm2, or Dnm3 proteins could inhibit WSSV replication in vivo. Overall, our research indicates the roles of host miRNAs in the enhancement of WSSV replication by regulating the host JAK/STAT pathway. PMID:27029712

  6. The role of RNA structure at 5' untranslated region in microRNA-mediated gene regulation.

    Science.gov (United States)

    Gu, Wanjun; Xu, Yuming; Xie, Xueying; Wang, Ting; Ko, Jae-Hong; Zhou, Tong

    2014-09-01

    Recent studies have suggested that the secondary structure of the 5' untranslated region (5' UTR) of messenger RNA (mRNA) is important for microRNA (miRNA)-mediated gene regulation in humans. mRNAs that are targeted by miRNA tend to have a higher degree of local secondary structure in their 5' UTR; however, the general role of the 5' UTR in miRNA-mediated gene regulation remains unknown. We systematically surveyed the secondary structure of 5' UTRs in both plant and animal species and found a universal trend of increased mRNA stability near the 5' cap in mRNAs that are regulated by miRNA in animals, but not in plants. Intra-genome comparison showed that gene expression level, GC content of the 5' UTR, number of miRNA target sites, and 5' UTR length may influence mRNA structure near the 5' cap. Our results suggest that the 5' UTR secondary structure performs multiple functions in regulating post-transcriptional processes. Although the local structure immediately upstream of the start codon is involved in translation initiation, RNA structure near the 5' cap site, rather than the structure of the full-length 5' UTR sequences, plays an important role in miRNA-mediated gene regulation.

  7. Proline biosynthesis genes and their regulation under salinity stress in the euryhaline copepod Tigriopus californicus.

    Science.gov (United States)

    Willett, Christopher S; Burton, Ronald S

    2002-08-01

    Diverse organisms regulate concentrations of intracellular organic osmolytes in response to changes in environmental salinity or desiccation. In marine crustaceans, accumulation of high concentrations of proline is a dominant component of response to hyperosmotic stress. In the euryhaline copepod Tigriopus californicus, synthesis of proline from its metabolic precursor glutamate is tightly regulated by changes in environmental salinity. Here, for the first time in a marine invertebrate, the genes responsible for this pathway have been cloned and characterized. The two proteins display the sequence features of homologous enzymes identified from other eukaryotes. One of the cloned genes, delta1-pyrroline-5-carboxylase reductase (P5CR), is demonstrated to have the reductase enzyme activity when expressed in proline-auxotroph bacteria, while the second, delta1-pyrroline-5-carboxylase synthase (P5CS), does not rescue proline-auxotroph bacteria. In contrast to results from higher plants, neither levels of P5CS nor P5CR mRNAs increase in response to salinity stress in T. californicus. Hence, regulation of proline synthesis during osmotic stress in T. californicus is likely mediated by some form of post-transcriptional regulation of either P5CS or P5CR. Understanding the regulation this pathway may elucidate the mechanisms limiting the salinity ranges of marine taxa.

  8. MicroRNA-24 Regulates Osteogenic Differentiation via Targeting T-Cell Factor-1

    Directory of Open Access Journals (Sweden)

    Weigong Zhao

    2015-05-01

    Full Text Available MicroRNAs (miRNAs have been reported to have diverse biological roles in regulating many biological processes, including osteogenic differentiation. In the present study, we identified that miR-24 was a critical regulator during osteogenic differentiation. We found that overexpression of miR-24 significantly inhibited osteogenic differentiation, which decreased alkaline phosphatase activity, matrix mineralization and the expression of osteogenic differentiation markers. In contrast, inhibition of miR-24 exhibited an opposite effect. Furthermore, we delineated that miR-24 regulates post-transcriptionals of T-cell factor-1 (Tcf-1 via targeting the 3'-untranslated region (UTR of Tcf-1 mRNA. MiR-24 was further found to regulate the protein expression of Tcf-1 in the murine osteoprogenitors cells and bone mesenchymal stem cells. Additionally, the positive effect of miR-24 suppression on osteoblast differentiation was apparently abrogated by Tcf-1 silencing. Taken together, our data suggest that miR-24 participates in osteogenic differentiation by targeting and regulating Tcf-1 expression in osteoblastic cells.

  9. SUMOylation of TARBP2 regulates miRNA/siRNA efficiency

    Science.gov (United States)

    Chen, Cheng; Zhu, Changhong; Huang, Jian; Zhao, Xian; Deng, Rong; Zhang, Hailong; Dou, Jinzhuo; Chen, Qin; Xu, Ming; Yuan, Haihua; Wang, Yanli; Yu, Jianxiu

    2015-01-01

    Small RNA-induced gene silencing is essential for post-transcriptional regulation of gene expression; however, it remains unclear how miRNA/siRNA efficiency is regulated. Here we show that TARBP2 is SUMOylated at K52, which can be enhanced by its phosphorylation. This modification can stabilize TARBP2 via repressing its K48-linked ubiquitination. We find that TARBP2 SUMOylation does not influence the overall production of mature miRNAs, but it regulates miRNA/siRNA efficiency. SUMOylated TARBP2 recruits Ago2 to constitute the RNA-induced silencing complex (RISC)-loading complex (RLC), and simultaneously promotes more pre-miRNAs to load into the RLC. Consequently, Ago2 is stabilized and miRNAs/siRNAs bound by TARBP2/Dicer is effectively transferred to Ago2. Thus, these processes lead to the formation of the effective RISC for RNA interference (RNAi). Collectively, our data suggest that SUMOylation of TARBP2 is required for regulating miRNA/siRNA efficiency, which is a general mechanism of miRNA/siRNA regulation. PMID:26582366

  10. Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs

    Directory of Open Access Journals (Sweden)

    Jonathan L. S. Esguerra

    2014-11-01

    Full Text Available Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs is termed “stimulus-secretion coupling.” Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D. The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.

  11. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

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    Stephanie Pitman

    2015-12-01

    Full Text Available The discovery of small noncoding regulatory RNAs (sRNAs in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

  12. Down-regulation of MiR-127 facilitates hepatocyte proliferation during rat liver regeneration.

    Directory of Open Access Journals (Sweden)

    Chuanyong Pan

    Full Text Available Liver regeneration (LR after partial hepatectomy (PH involves the proliferation and apoptosis of hepatocytes, and microRNAs have been shown to post-transcriptionally regulate genes involved in the regulation of these processes. To explore the role of miR-127 during LR, the expression patterns of miR-127 and its related proteins were investigated. MiR-127 was introduced into a rat liver cell line to examine its effects on the potential target genes Bcl6 and Setd8, and functional studies were undertaken. We discovered that miR-127 was down-regulated and inversely correlated with the expression of Bcl6 and Setd8 at 24 hours after PH, a time at which hypermethylation of the promoter region of the miR-127 gene was detected. Furthermore, in BRL-3A rat liver cells, we observed that overexpression of miR-127 significantly suppressed cell growth and directly inhibited the expression of Bcl6 and Setd8. The results suggest that down-regulation of miR-127 may be due to the rapid methylation of its promoter during the first 24 h after PH, and this event facilitates hepatocyte proliferation by releasing Bcl6 and Setd8. These findings support a miRNA-mediated negative regulation pattern in LR and implicate an anti-proliferative role for miR-127 in liver cells.

  13. Reciprocal regulation of very low density lipoprotein receptors (VLDLRs) in neurons by brain-derived neurotrophic factor (BDNF) and Reelin: involvement of the E3 ligase Mylip/Idol.

    Science.gov (United States)

    Do, Hai Thi; Bruelle, Céline; Tselykh, Timofey; Jalonen, Pilvi; Korhonen, Laura; Lindholm, Dan

    2013-10-11

    BDNF positively influences various aspects of neuronal migration, maturation, and survival in the developing brain. Reelin in turn mediates inhibitory signals to migrating neuroblasts, which is crucial for brain development. The interplay between BDNF and Reelin signaling in neurodevelopment is not fully understood. We show here that BDNF increased the levels of the Reelin receptor (VLDL receptor (VLDLR)) in hippocampal neurons by increasing gene expression. In contrast, Reelin decreased VLDLRs, which was accompanied by an increase in the levels of the E3 ligase Mylip/Idol in neurons. Down-regulation of Mylip/Idol using shRNAs abrogated the decrease in VLDLRs induced by Reelin. These results show that VLDLRs are tightly regulated in hippocampal neurons by both transcriptional and post-transcriptional mechanisms. The regulation of VLDLR by BDNF and Reelin may affect the migration of neurons and contribute to neurodevelopmental disorders in the nervous system.

  14. The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA.

    Science.gov (United States)

    López de Silanes, Isabel; Gorospe, Myriam; Taniguchi, Hiroaki; Abdelmohsen, Kotb; Srikantan, Subramanya; Alaminos, Miguel; Berdasco, María; Urdinguio, Rocío G; Fraga, Mario F; Jacinto, Filipe V; Esteller, Manel

    2009-05-01

    The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

  15. Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.

    Directory of Open Access Journals (Sweden)

    Stuart K Archer

    2009-08-01

    Full Text Available Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9--LIGKA, PNT1 and PNT2--were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3' UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3' UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.

  16. MicroRNA as type I interferon-regulated transcripts and modulators of the innate immune response

    Directory of Open Access Journals (Sweden)

    Samuel C Forster

    2015-07-01

    Full Text Available Type I interferons (IFNs are an important family of cytokines that regulate innate and adaptive immune responses to pathogens, in cancer and inflammatory diseases. While the regulation and role of protein-coding genes involved in these responses are well characterized, the role of non-coding microRNAs in the IFN responses is less developed. We review the emerging picture of microRNA regulation of the IFN response at the transcriptional and post-transcriptional level. This response forms an important regulatory loop, several microRNAs target transcripts encoding components at many steps of the type I IFN response, both production and action, at the receptor, signaling, transcription factor and regulated gene level. Not only do IFNs regulate positive signaling molecules, but also negative regulators such as SOCS1. In total, 36 microRNA are reported as IFN regulated. Given this apparent multipronged targeting of the IFN response by microRNAs and their well characterized capacity to buffer responses in other situations, the prospects of improved sequencing and microRNA targeting technologies will facilitate the elucidation of the broader regulatory networks of microRNA in this important biological context, and their therapeutic and diagnostic potential.

  17. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  18. Regulation of MicroRNA Biogenesis: A miRiad of mechanisms

    Directory of Open Access Journals (Sweden)

    Davis Brandi N

    2009-08-01

    Full Text Available Abstract microRNAs are small, non-coding RNAs that influence diverse biological functions through the repression of target genes during normal development and pathological responses. Widespread use of microRNA arrays to profile microRNA expression has indicated that the levels of many microRNAs are altered during development and disease. These findings have prompted a great deal of investigation into the mechanism and function of microRNA-mediated repression. However, the mechanisms which govern the regulation of microRNA biogenesis and activity are just beginning to be uncovered. Following transcription, mature microRNA are generated through a series of coordinated processing events mediated by large protein complexes. It is increasingly clear that microRNA biogenesis does not proceed in a 'one-size-fits-all' manner. Rather, individual classes of microRNAs are differentially regulated through the association of regulatory factors with the core microRNA biogenesis machinery. Here, we review the regulation of microRNA biogenesis and activity, with particular focus on mechanisms of post-transcriptional control. Further understanding of the regulation of microRNA biogenesis and activity will undoubtedly provide important insights into normal development as well as pathological conditions such as cardiovascular disease and cancer.

  19. Hypoxia: A Master Regulator of MicroRNA Biogenesis and Activity

    Science.gov (United States)

    Nallamshetty, Shriram; Chan, Stephen Y.; Loscalzo, Joseph

    2013-01-01

    Hypoxia, or low oxygen tension, is a unique environmental stress that induces global changes in a complex regulatory network of transcription factors and signaling proteins in order to coordinate cellular adaptations in metabolism, proliferation, DNA repair, and apoptosis. Several lines of evidence now establish microRNAs (miRNAs), which are short non-coding RNAs that regulate gene expression through post-transcriptional mechanisms, as key elements in this response to hypoxia. Oxygen deprivation induces a distinct shift in a specific group of miRNAs, termed hypoxamirs, and emerging evidence indicates that hypoxia regulates several facets of hypoxamir transcription, maturation, and function. Transcription factors such as hypoxia-inducible factor (HIF) are upregulated under conditions of low oxygen availability and directly activate the transcription of a subset of hypoxamirs. Conversely, hypoxia selectively represses other hypoxamirs through less well characterized mechanisms. In addition, oxygen deprivation has been directly implicated in epigenetic modifications such as DNA demethylation that control specific miRNA transcription. Finally, hypoxia also modulates the activity of key proteins that control posttranscriptional events in the maturation and activity of miRNAs. Collectively, these findings establish hypoxia as an important proximal regulator of miRNA biogenesis and function. It will be important for future studies to address the relative contributions of transcriptional and posttranscriptional events in the regulation of specific hypoxamirs and how such miRNAs are coordinated order to integrate into the complex hierarchical regulatory network induced by hypoxia. PMID:23712003

  20. Gravity-regulated gene expression in Arabidopsis thaliana

    Science.gov (United States)

    Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

    Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

  1. bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice

    Science.gov (United States)

    Ranjan, Rajeev; Khurana, Reema; Malik, Naveen; Badoni, Saurabh; Parida, Swarup K.; Kapoor, Sanjay; Tyagi, Akhilesh K.

    2017-01-01

    Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142OEplants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes. PMID:28262713

  2. Transforming growth factor-β3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

    Science.gov (United States)

    Zhang, Xu; Lui, Wing-Yee

    2015-06-01

    Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-β) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-β3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-β3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-β3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-β3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-β3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-β3-induced JAM-B protein degradation. In addition, TGF-β3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-β3 promotes the degradation of JAM-B transcript and TGF-β3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-β3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES.

  3. Fungal virulence and development is regulated by alternative pre-mRNA 3'end processing in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Marina Franceschetti

    2011-12-01

    Full Text Available RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3'UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3'end processing mechanisms exclusive to the fungal kingdom.

  4. Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

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    Enrique Álvarez

    Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

  5. Genomic Analyses of Sperm Fate Regulator Targets Reveal a Common Set of Oogenic mRNAs in Caenorhabditis elegans.

    Science.gov (United States)

    Noble, Daniel C; Aoki, Scott T; Ortiz, Marco A; Kim, Kyung Won; Verheyden, Jamie M; Kimble, Judith

    2016-01-01

    Germ cell specification as sperm or oocyte is an ancient cell fate decision, but its molecular regulation is poorly understood. In Caenorhabditis elegans, the FOG-1 and FOG-3 proteins behave genetically as terminal regulators of sperm fate specification. Both are homologous to well-established RNA regulators, suggesting that FOG-1 and FOG-3 specify the sperm fate post-transcriptionally. We predicted that FOG-1 and FOG-3, as terminal regulators of the sperm fate, might regulate a battery of gamete-specific differentiation genes. Here we test that prediction by exploring on a genomic scale the messenger RNAs (mRNAs) associated with FOG-1 and FOG-3. Immunoprecipitation of the proteins and their associated mRNAs from spermatogenic germlines identifies 81 FOG-1 and 722 FOG-3 putative targets. Importantly, almost all FOG-1 targets are also FOG-3 targets, and these common targets are strongly biased for oogenic mRNAs. The discovery of common target mRNAs suggested that FOG-1 and FOG-3 work together. Consistent with that idea, we find that FOG-1 and FOG-3 proteins co-immunoprecipitate from both intact nematodes and mammalian tissue culture cells and that they colocalize in germ cells. Taking our results together, we propose a model in which FOG-1 and FOG-3 work in a complex to repress oogenic transcripts and thereby promote the sperm fate.

  6. Computational prediction of microRNAs from Toxoplasma gondii potentially regulating the hosts' gene expression.

    Science.gov (United States)

    Saçar, Müşerref Duygu; Bağcı, Caner; Allmer, Jens

    2014-10-01

    MicroRNAs (miRNAs) were discovered two decades ago, yet there is still a great need for further studies elucidating their genesis and targeting in different phyla. Since experimental discovery and validation of miRNAs is difficult, computational predictions are indispensable and today most computational approaches employ machine learning. Toxoplasma gondii, a parasite residing within the cells of its hosts like human, uses miRNAs for its post-transcriptional gene regulation. It may also regulate its hosts' gene expression, which has been shown in brain cancer. Since previous studies have shown that overexpressed miRNAs within the host are causal for disease onset, we hypothesized that T. gondii could export miRNAs into its host cell. We computationally predicted all hairpins from the genome of T. gondii and used mouse and human models to filter possible candidates. These were then further compared to known miRNAs in human and rodents and their expression was examined for T. gondii grown in mouse and human hosts, respectively. We found that among the millions of potential hairpins in T. gondii, only a few thousand pass filtering using a human or mouse model and that even fewer of those are expressed. Since they are expressed and differentially expressed in rodents and human, we suggest that there is a chance that T. gondii may export miRNAs into its hosts for direct regulation.

  7. Roles of Calcium Regulating MicroRNAs in Cardiac Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Eunhyun Choi

    2014-09-01

    Full Text Available Cardiac Ca2+ cycling and signaling are closely associated with cardiac function. Changes in cellular Ca2+ homeostasis may lead to aberrant cardiac rhythm and may play a critical role in the pathogenesis of cardiac diseases, due to their exacerbation of heart failure. MicroRNAs (miRNAs play a key role in the regulation of gene expression at the post-transcriptional level and participate in regulating diverse biological processes. The emerging evidence indicates that the expression profiles of miRNAs vary among human diseases, including cardiovascular diseases. Cardiac Ca2+-handling and signaling proteins are also regulated by miRNAs. Given the relationship between cardiac Ca2+ homeostasis and signaling and miRNA, Ca2+-related miRNAs may serve as therapeutic targets during the treatment of heart failure. In this review, we summarize the knowledge currently available regarding the role of Ca2+ in cardiac function, as well as changes in Ca2+ cycling and homeostasis and the handling of these processes by miRNAs during cardiac ischemia-reperfusion injury.

  8. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

    Science.gov (United States)

    Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

    2011-02-15

    At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

  9. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells.

    Science.gov (United States)

    Ortega, Alvaro D; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  10. Differential expression of speckled POZ protein, SPOP: Putative regulation by miR-145

    Indian Academy of Sciences (India)

    Chiu-Jung Huang; Hsing-Yu Chen; Wan-Yi Lin; Kongbung Choo

    2014-06-01

    The speckle POZ protein, SPOP, is an adaptor of the Cul3-based ubiquitination process, and has been implicated in the carcinogenesis process. Despite recent elucidation of biological functions, regulation of SPOP gene expression has not been reported. In this study, the mRNA levels of the mouse SPOP (mSPOP) gene were first shown to vary noticeably in different tissues. However, the SPOP protein was detected in high abundance only in Purkinje cells of the cerebellum and seminiferous tubule of the testis, echoing previous reports of involvement of ubiquitination in neuron cells and in spermatogenesis. In other mouse tissues and human cancer cell lines analysed, only low SPOP protein levels were detected. The 3′-untranslated regions of both the mSPOP and human SPOP transcripts harbor a conserved putative miR-145 binding site (BS). In some tissues and cell lines, miR-145 and SPOP protein levels were in an inverse relationship suggesting miR-145 regulation. Luciferase assays of deletion and point mutation constructs of the miR-145 BS, and miR-145 induction by serum starvation that resulted in reduced endogenous SPOP levels provided further evidence that miR-145 is likely involved in post-transcriptional regulation of SPOP expression in selected tissues, and possibly with the participation of other miRNA species.

  11. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  12. Computational Prediction of MicroRNAs from Toxoplasma gondii Potentially Regulating the Hosts’ Gene Expression

    Institute of Scientific and Technical Information of China (English)

    Muserref Duygu Sacar; Caner Bagc; Jens Allmer

    2014-01-01

    MicroRNAs (miRNAs) were discovered two decades ago, yet there is still a great need for further studies elucidating their genesis and targeting in different phyla. Since experimental discovery and validation of miRNAs is difficult, computational predictions are indispensable and today most computational approaches employ machine learning. Toxoplasma gondii, a parasite residing within the cells of its hosts like human, uses miRNAs for its post-transcriptional gene reg-ulation. It may also regulate its hosts’ gene expression, which has been shown in brain cancer. Since previous studies have shown that overexpressed miRNAs within the host are causal for disease onset, we hypothesized that T. gondii could export miRNAs into its host cell. We computationally predicted all hairpins from the genome of T. gondii and used mouse and human models to filter possible candidates. These were then further compared to known miRNAs in human and rodents and their expression was examined for T. gondii grown in mouse and human hosts, respectively. We found that among the millions of potential hairpins in T. gondii, only a few thousand pass filtering using a human or mouse model and that even fewer of those are expressed. Since they are expressed and differentially expressed in rodents and human, we suggest that there is a chance that T. gondii may export miRNAs into its hosts for direct regulation.

  13. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

    Science.gov (United States)

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria

    2011-03-01

    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  14. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Álvaro D. Ortega

    2014-11-01

    Full Text Available Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of ‘intact’ intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  15. In silico identification and in vivo validation of miR-495 as a novel regulator of motivation for cocaine that targets multiple addiction-related networks in the nucleus accumbens.

    Science.gov (United States)

    Bastle, R M; Oliver, R J; Gardiner, A S; Pentkowski, N S; Bolognani, F; Allan, A M; Chaudhury, T; St Peter, M; Galles, N; Smith, C; Neisewander, J L; Perrone-Bizzozero, N I

    2017-01-03

    MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase for Addiction Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3'UTRs of Bdnf, Camk2a and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that have a role in modulating motivation for cocaine.Molecular Psychiatry advance online publication, 3 January 2017; doi:10.1038/mp.2016.238.

  16. Genetic interaction of two abscisic acid signaling regulators, HY5 and FIERY1, in mediating lateral root formation

    KAUST Repository

    Chen, Hao

    2011-01-01

    Root architecture is continuously shaped in a manner that helps plants to better adapt to the environment. Gene regulation at the transcriptional or post-transcriptional levels largely controls this environmental response. Recently, RNA silencing has emerged as an important player in gene regulation and is involved in many aspects of plant development, including lateral root formation. In a recent study, we found that FIERY1, a bifunctional abiotic stress and abscisic acid (ABA) signaling regulator and an endogenous RNA silencing suppressor, mediates auxin response during lateral root formation in Arabidopsis. We proposed that FRY1 regulates lateral root development through its activity on adenosine 3,5-bisphosphate (PAP), a strong inhibitor of exoribonucleases (XRNs). Interestingly, some of the phenotypes of fry1, such as enhanced response to light in repressing hypocotyl elongation and hypersensitivity to ABA in lateral root growth, are opposite to those of another light- and ABA-signaling mutant, hy5. Here we analyzed the hy5 fry1 double mutant for root and hypocotyl growth. We found that the hy5 mutation can suppress the enhanced light sensitivity in fry1 hypocotyl elongation and restore the lateral root formation. The genetic interaction between HY5 and FRY1 indicates that HY5 and FRY1 may act in overlapping pathways that mediate light signaling and lateral root development. © 2011 Landes Bioscience.

  17. MiR-146 and miR-125 in the regulation of innate immunity and inflammation

    Science.gov (United States)

    Lee, Hye-Mi; Kim, Tae Sung; Jo, Eun-Kyeong

    2016-01-01

    Innate immune responses are primary, relatively limited, and specific responses to numerous pathogens and toxic molecules. Protein expression involved in these innate responses must be tightly regulated at both transcriptional level and post-transcriptional level to avoid the development of excessive inflammation that can be potentially harmful to the host. MicroRNAs are small noncoding RNAs (∼22 nucleotides [nts]) that participate in the regulation of numerous physiological responses by targeting specific messenger RNAs to suppress their translation. Recent work has shown that several negative regulators of transcription including microRNAs play important roles in inhibiting the exacerbation of inflammatory responses and in the maintenance of immunological homeostasis. This emerging research area will provide new insights on how microRNAs regulate innate immune signaling. It might show that dysregulation of microRNA synthesis is associated with the pathogenesis of inflammatory and infectious diseases. In this review, we focused on miR-146 and miR-125 and described the roles these miRNAs in modulating innate immune signaling. These microRNAs can control inflammatory responses and the outcomes of pathogenic infections. [BMB Reports 2016; 49(6): 311-318] PMID:26996343

  18. Angiogenesis-regulating microRNAs and Ischemic Stroke.

    Science.gov (United States)

    Yin, Ke-Jie; Hamblin, Milton; Chen, Y Eugene

    2015-01-01

    Stroke is a leading cause of death and disability worldwide. Ischemic stroke is the dominant subtype of stroke and results from focal cerebral ischemia due to occlusion of major cerebral arteries. Thus, the restoration or improvement of reduced regional cerebral blood supply in a timely manner is very critical for improving stroke outcomes and poststroke functional recovery. The recovery from ischemic stroke largely relies on appropriate restoration of blood flow via angiogenesis. Newly formed vessels would allow increased cerebral blood flow, thus increasing the amount of oxygen and nutrients delivered to affected brain tissue. Angiogenesis is strictly controlled by many key angiogenic factors in the central nervous system, and these molecules have been well-documented to play an important role in the development of angiogenesis in response to various pathological conditions. Promoting angiogenesis via various approaches that target angiogenic factors appears to be a useful treatment for experimental ischemic stroke. Most recently, microRNAs (miRs) have been identified as negative regulators of gene expression in a post-transcriptional manner. Accumulating studies have demonstrated that miRs are essential determinants of vascular endothelial cell biology/angiogenesis as well as contributors to stroke pathogenesis. In this review, we summarize the knowledge of stroke-associated angiogenic modulators, as well as the role and molecular mechanisms of stroke-associated miRs with a focus on angiogenesis-regulating miRs. Moreover, we further discuss their potential impact on miR-based therapeutics in stroke through targeting and enhancing post-ischemic angiogenesis.

  19. A Csr-type regulatory system, including small non-coding RNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis through RovM.

    Science.gov (United States)

    Heroven, Ann Kathrin; Böhme, Katja; Rohde, Manfred; Dersch, Petra

    2008-06-01

    The MarR-type regulator RovA controls expression of virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic strategy to discover components that influence rovA expression, we identified new regulatory factors with homology to components of the carbon storage regulator system (Csr). We showed that overexpression of a CsrB- or a CsrC-type RNA activates rovA, whereas a CsrA-like protein represses RovA synthesis. We further demonstrate that influence of the Csr system on rovA is indirect and occurs through control of the LysR regulator RovM, which inhibits rovA transcription. The CsrA protein had also a major influence on the motility of Yersinia, which was independent of RovM. The CsrB and CsrC RNAs are differentially expressed in Yersinia. CsrC is highly induced in complex but not in minimal media, indicating that medium-dependent rovM expression is mediated through CsrC. CsrB synthesis is generally very low. However, overexpression of the response regulator UvrY was found to activate CsrB production, which in turn represses CsrC synthesis independent of the growth medium. In summary, the post-transcriptional Csr-type components were shown to be key regulators in the co-ordinated environmental control of physiological processes and virulence factors, which are crucial for the initiation of Yersinia infections.

  20. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

    Energy Technology Data Exchange (ETDEWEB)

    Eisenstein, R.S.; Rosen, J.M.

    1988-08-01

    The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNA was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.

  1. Differences in expression, actions and cocaine regulation of two isoforms for the brain transcriptional regulator NAC1.

    Science.gov (United States)

    Korutla, L; Wang, P J; Lewis, D M; Neustadter, J H; Stromberg, M F; Mackler, S A

    2002-01-01

    BTB/POZ proteins can influence the cell cycle and contribute to oncogenesis. Many family members are present in the mammalian CNS. Previous work demonstrated elevated NAC1 mRNA levels in the rat nucleus accumbens in response to cocaine. NAC1 acts like other BTB/POZ proteins that regulate transcription but is unusual because of the absence of identifiable DNA binding domains. cDNAs were isolated encoding two NAC1 isoforms differing by only 27 amino acids (the longer isoform contains 514 amino acids). The mRNAs for both isoforms were simultaneously expressed throughout the rat brain and peripheral tissues. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that the mRNA of the longer isoform was more abundant than the mRNA of the shorter isoform. Western blot analysis demonstrated a similar unequal distribution between the isoforms in the CNS. The longer isoform was the more abundant of the two NAC1 proteins and the ratio between them differed throughout the rat brain. The shorter isoform was not detected in most of the examined peripheral tissues, suggesting differences from the CNS in post-transcriptional processing. Both isoforms repressed transcription in H293T cells using a Gal4-luciferase reporter system. However, the shorter isoform did not repress transcription as effectively as the longer isoform. Transfection of different ratios for both isoforms, in order to replicate the relative amounts observed throughout the CNS, supported an interaction between the isoforms. The net effect on transcriptional repression was determined by the ratio of the two NAC1 isoforms. Each isoform exhibited the subnuclear localization that is characteristic of many BTB/POZ proteins. A rapid and transient increase in the level of the shorter isoform occurred in the nucleus accumbens 2 h following a single i.p. cocaine injection. We conclude that the two isoforms of NAC1 may differentially affect neuronal functions, including the regulation of

  2. Identification of critical paralog groups with indispensable roles in the regulation of signaling flow.

    Science.gov (United States)

    Modos, Dezso; Brooks, Johanne; Fazekas, David; Ari, Eszter; Vellai, Tibor; Csermely, Peter; Korcsmaros, Tamas; Lenti, Katalin

    2016-12-06

    Extensive cross-talk between signaling pathways is required to integrate the myriad of extracellular signal combinations at the cellular level. Gene duplication events may lead to the emergence of novel functions, leaving groups of similar genes - termed paralogs - in the genome. To distinguish critical paralog groups (CPGs) from other paralogs in human signaling networks, we developed a signaling network-based method using cross-talk annotation and tissue-specific signaling flow analysis. 75 CPGs were found with higher degree, betweenness centrality, closeness, and 'bowtieness' when compared to other paralogs or other proteins in the signaling network. CPGs had higher diversity in all these measures, with more varied biological functions and more specific post-transcriptional regulation than non-critical paralog groups (non-CPG). Using TGF-beta, Notch and MAPK pathways as examples, SMAD2/3, NOTCH1/2/3 and MEK3/6-p38 CPGs were found to regulate the signaling flow of their respective pathways. Additionally, CPGs showed a higher mutation rate in both inherited diseases and cancer, and were enriched in drug targets. In conclusion, the results revealed two distinct types of paralog groups in the signaling network: CPGs and non-CPGs. Thus highlighting the importance of CPGs as compared to non-CPGs in drug discovery and disease pathogenesis.

  3. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    Science.gov (United States)

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  4. dPORE-miRNA: Polymorphic regulation of microRNA genes

    KAUST Repository

    Schmeier, Sebastian

    2011-02-04

    Background: MicroRNAs (miRNAs) are short non-coding RNA molecules that act as post-transcriptional regulators and affect the regulation of protein-coding genes. Mostly transcribed by PolII, miRNA genes are regulated at the transcriptional level similarly to protein-coding genes. In this study we focus on human miRNAs. These miRNAs are involved in a variety of pathways and can affect many diseases. Our interest is on possible deregulation of the transcription initiation of the miRNA encoding genes, which is facilitated by variations in the genomic sequence of transcriptional control regions (promoters). Methodology: Our aim is to provide an online resource to facilitate the investigation of the potential effects of single nucleotide polymorphisms (SNPs) on miRNA gene regulation. We analyzed SNPs overlapped with predicted transcription factor binding sites (TFBSs) in promoters of miRNA genes. We also accounted for the creation of novel TFBSs due to polymorphisms not present in the reference genome. The resulting changes in the original TFBSs and potential creation of new TFBSs were incorporated into the Dragon Database of Polymorphic Regulation of miRNA genes (dPORE-miRNA). Conclusions: The dPORE-miRNA database enables researchers to explore potential effects of SNPs on the regulation of miRNAs. dPORE-miRNA can be interrogated with regards to: a/miRNAs (their targets, or involvement in diseases, or biological pathways), b/SNPs, or c/transcription factors. dPORE-miRNA can be accessed at http://cbrc.kaust.edu.sa/dpore and http://apps.sanbi.ac.za/dpore/. Its use is free for academic and non-profit users. © 2011 Schmeier et al.

  5. MicroRNA-383 regulates the apoptosis of tumor cells through targeting Gadd45g.

    Directory of Open Access Journals (Sweden)

    Lei Zhao

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of small non-coding single-stranded RNA molecules that inhibit gene expression at post-transcriptional level. Gadd45g (growth arrest and DNA-damage-inducible 45 gamma is a stress-response protein, which has been implicated in several biological processes, including DNA repair, the cell cycle and cell differentiation. RESULTS: In this work, we found that miR-383 is a negative regulator of Gadd45g. Forced expression of miR-383 decreased the expression of Gadd45g through binding to the 3' untranslated region (3'-UTR, whereas inhibition of miR-383 increased Gadd45g expression. The presence of miR-383 increased the cellular sensitivity to DNA damage in breast cancer cells, which was rescued by ectopic expression of Gadd45g without the 3'-UTR. miR-383 also regulates the expression of Gadd45g in embryonic stem (ES cells, but not their apoptosis under genotoxic stress. miR-383 was further showed to negatively regulate ES cell differentiation via targeting Gadd45g, which subsequently modulates the pluripotency-associated genes. Taken together, our study demonstrates that miR-383 is a negative regulator of Gadd45g in both tumor cells and ES cells, however, has distinct function in regulating cell apoptosis. miR-383 may be used as antineoplastic agents in cancer chemotherapy. CONCLUSION: We demonstrate for the first time that miR-383 can specifically regulates the expression of Gadd45g by directly targeting to the 3-UTR region of Gadd45g mRNA, a regulatory process conserved in human tumor cells and mouse embryonic stem cells. These two compotents can be potentially used as antineoplastic agents in cancer chemotherapy.

  6. Involvement of cyclin K posttranscriptional regulation in the formation of Artemia diapause cysts.

    Directory of Open Access Journals (Sweden)

    Yang Zhao

    Full Text Available BACKGROUND: Artemia eggs tend to develop ovoviviparously to yield nauplius larvae in good rearing conditions; while under adverse situations, they tend to develop oviparously and encysted diapause embryos are formed instead. However, the intrinsic mechanisms regulating this process are not well understood. PRINCIPAL FINDING: This study has characterized the function of cyclin K, a regulatory subunit of the positive transcription elongation factor b (P-TEFb in the two different developmental pathways of Artemia. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2 in the C-terminal domain (CTD of the largest subunit (Rpb1 of RNA polymerase II (RNAP II. Interestingly, the cyclin K transcript levels remained constant during this process. In vitro translation data indicated that the template activity of cyclin K mRNA stored in the postdiapause cyst was repressed. In addition, in vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser2 of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK survival signaling pathway. CONCLUSIONS/SIGNIFICANCE: Taken together, these findings reveal a role for cyclin K in regulating RNAP II activity during diapause embryo development, which involves the post-transcriptional regulation of cyclin K. In addition, a further role was identified for cyclin K in regulating the control of cell survival during embryogenesis through ERK signaling pathways.

  7. microRNA as a new agent for regulating neuronal glutathione synthesis and metabolism

    Directory of Open Access Journals (Sweden)

    Chisato Kinoshita

    2015-04-01

    Full Text Available microRNA (miRNA is a small non-coding RNA molecule that plays a role in the post-transcriptional regulation of gene expression. Recent evidence shows that miRNAs are involved in various diseases, including neurodegenerative diseases (NDs such as: Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Amyotrophic lateral sclerosisand multiple system atrophy (MSA. The initiation and progression of NDs is generally considered to be induced by oxidative stress arising from an imbalance of oxidants and antioxidants. One of the most important antioxidants against oxidative stress is glutathione (GSH, which is a tripeptide composed of cysteine, glutamate and glycine. Among these precursor amino acids, cysteine is the determinant of neuronal GSH synthesis. Cysteine uptake in the neurons is mostly mediated by excitatory amino acid carrier 1 (EAAC1, a member of the sodium-dependent excitatory amino acid transporters. Interestingly, it has been reported that one miRNA, miR-96-5p, regulates the neuroprotective effect of GSH by directly regulating EAAC1 expression. Furthermore, the expressions of miR-96-5p and its target EAAC1 are specifically deregulated in the brains of patients with MSA, suggesting that deregulated miR-96-5p induces MSA via EAAC1 down-regulation. Since miR-96-5p regulation of EAAC1 expression and GSH level is indicated to be under circadian control, a greater understanding of rhythmic miRNA regulation could lead to the use of miRNA in chronotherapy for ND. In this review, we focus on the role of miRNA in the mechanism of GSH synthesis and metabolism; particularly with respect to a critical transport system of its rate-limiting substrate via EAAC1, as well as on the implications and chronotherapeutic potential of miRNA for NDs.

  8. miRNA in the regulation of skeletal muscle adaptation to acute endurance exercise in C57Bl/6J male mice.

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    Adeel Safdar

    Full Text Available MicroRNAs (miRNAs are evolutionarily conserved small non-coding RNA species involved in post-transcriptional gene regulation. In vitro studies have identified a small number of skeletal muscle-specific miRNAs which play a crucial role in myoblast proliferation and differentiation. In skeletal muscle, an acute bout of endurance exercise results in the up-regulation of transcriptional networks that regulate mitochondrial biogenesis, glucose and fatty acid metabolism, and skeletal muscle remodelling. The purpose of this study was to assess the expressional profile of targeted miRNA species following an acute bout of endurance exercise and to determine relationships with previously established endurance exercise responsive transcriptional networks. C57Bl/6J wild-type male mice (N = 7/group were randomly assigned to either sedentary or forced-endurance exercise (treadmill run @ 15 m/min for 90 min group. The endurance exercise group was sacrificed three hours following a single bout of exercise. The expression of miR- 181, 1, 133, 23, and 107, all of which have been predicted to regulate transcription factors and co-activators involved in the adaptive response to exercise, was measured in quadriceps femoris muscle. Endurance exercise significantly increased the expression of miR-181, miR-1, and miR-107 by 37%, 40%, and 56%, respectively, and reduced miR-23 expression by 84% (Pregulator of PGC-1alpha was consistent with increased expression of PGC-1alpha mRNA and protein along with several downstream targets of PGC-1alpha including ALAS, CS, and cytochrome c mRNA. PDK4 protein content remains unaltered despite an increase in its putative negative regulator, miR-107, and PDK4 mRNA expression. mRNA expression of miRNA processing machinery (Drosha, Dicer, and DGCR8 remained unchanged. We conclude that miRNA-mediated post-transcriptional

  9. Autophagy Regulatory Network - a systems-level bioinformatics resource for studying the mechanism and regulation of autophagy.

    Science.gov (United States)

    Türei, Dénes; Földvári-Nagy, László; Fazekas, Dávid; Módos, Dezső; Kubisch, János; Kadlecsik, Tamás; Demeter, Amanda; Lenti, Katalin; Csermely, Péter; Vellai, Tibor; Korcsmáros, Tamás

    2015-01-01

    Autophagy is a complex cellular process having multiple roles, depending on tissue, physiological, or pathological conditions. Major post-translational regulators of autophagy are well known, however, they have not yet been collected comprehensively. The precise and context-dependent regulation of autophagy necessitates additional regulators, including transcriptional and post-transcriptional components that are listed in various datasets. Prompted by the lack of systems-level autophagy-related information, we manually collected the literature and integrated external resources to gain a high coverage autophagy database. We developed an online resource, Autophagy Regulatory Network (ARN; http://autophagy-regulation.org), to provide an integrated and systems-level database for autophagy research. ARN contains manually curated, imported, and predicted interactions of autophagy components (1,485 proteins with 4,013 interactions) in humans. We listed 413 transcription factors and 386 miRNAs that could regulate autophagy components or their protein regulators. We also connected the above-mentioned autophagy components and regulators with signaling pathways from the SignaLink 2 resource. The user-friendly website of ARN allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. ARN has the potential to facilitate the experimental validation of novel autophagy components and regulators. In addition, ARN helps the investigation of transcription factors, miRNAs and signaling pathways implicated in the control of the autophagic pathway. The list of such known and predicted regulators could be important in pharmacological attempts against cancer and neurodegenerative diseases.

  10. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1.

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    Qinna Cui

    Full Text Available Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1 and phz2 (phzA2B2C2D2E2F2G2] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1.

  11. An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.

    Science.gov (United States)

    Robertson, Kevin A; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J; Enright, Anton J; Yamamoto, Mami; Pradeepa, Madapura M; Lennox, Kimberly A; Behlke, Mark A; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

    2016-03-01

    In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway.

  12. Regulation of Aicda expression and AID activity.

    Science.gov (United States)

    Zan, Hong; Casali, Paolo

    2013-03-01

    Activation-induced cytidine deaminase (AID) is expressed in a B cell differentiation stage-specific fashion and is essential for immunoglobulin (Ig) gene class switch DNA recombination (CSR) and somatic hypermutation (SHM). CSR and SHM play a central role in the maturation of antibody and autoantibody responses. AID displays a mutagenic activity by catalyzing targeted deamination of deoxycytidine (dC) residues in DNA resulting in dU:dG mismatches, which are processed into point-mutations in SHM or double-strand breaks (DSBs) in CSR. Although AID specifically targets the Ig gene loci (IgH, Igκ and Igλ), it can also home into a wide array of non-Ig genes in B-and non-B-cell backgrounds. Aberrant expression of AID is associated with multiple diseases such as allergy, inflammation, autoimmunity and cancer. In autoimmune systemic lupus erythematosus, dysregulated AID expression underpins increased CSR, SHM and autoantibody production. As a potent mutator, AID is under stringent transcriptional, post-transcriptional and post-translational regulation. AID is also regulated in its targeting and enzymatic function. In resting naïve or memory B cells, AID transcripts and protein are undetectable. These, however, are readily and significantly up-regulated in B cells induced to undergo CSR and/or SHM. Transcription factors, such as HoxC4 and NF-κB, which are up-regulated in a B cell lineage-and/or differentiation stage-specific manner, regulate the induction of AID. HoxC4 induces AID expression by directly binding to the AID gene promoter through an evolutionarily conserved 5'-ATTT-3' motif. HoxC4 is induced by the same stimuli that induce AID and CSR. It is further up-regulated by estrogen through three estrogen responsive elements in its promoter region. The targeting of AID to switch (S) regions is mediated by 14-3-3 adaptor proteins, which specifically bind to 5'-AGCT-3' repeats that are exist at high frequency in S region cores. Like HoxC4, 14-3-3 adaptors are induced

  13. Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events.

    Science.gov (United States)

    Belostotsky, Dmitry A; Rose, Alan B

    2005-07-01

    The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.

  14. Identification of Post-Transcriptional Modulators of Breast Cancer Transcription Factor Activity Using MINDy

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    Campbell, Thomas M.; Castro, Mauro A. A.; Ponder, Bruce A. J.

    2016-01-01

    We have recently identified transcription factors (TFs) that are key drivers of breast cancer risk. To better understand the pathways or sub-networks in which these TFs mediate their function we sought to identify upstream modulators of their activity. We applied the MINDy (Modulator Inference by Network Dynamics) algorithm to four TFs (ESR1, FOXA1, GATA3 and SPDEF) that are key drivers of estrogen receptor-positive (ER+) breast cancer risk, as well as cancer progression. Our computational analysis identified over 500 potential modulators. We assayed 189 of these and identified 55 genes with functional characteristics that were consistent with a role as TF modulators. In the future, the identified modulators may be tested as potential therapeutic targets, able to alter the activity of TFs that are critical in the development of breast cancer. PMID:27997592

  15. Glucose-Regulated Phosphorylation of the PUF Protein Puf3 Regulates the Translational Fate of Its Bound mRNAs and Association with RNA Granules

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    Chien-Der Lee

    2015-06-01

    Full Text Available PUF proteins are post-transcriptional regulators that bind to the 3′ UTRs of mRNA transcripts. Herein, we show how a yeast PUF protein, Puf3p, responds to glucose availability to switch the fate of its bound transcripts that encode proteins required for mitochondrial biogenesis. Upon glucose depletion, Puf3p becomes heavily phosphorylated within its N-terminal region of low complexity, associates with polysomes, and promotes translation of its target mRNAs. Such nutrient-responsive phosphorylation toggles the activity of Puf3p to promote either degradation or translation of these mRNAs according to the needs of the cell. Moreover, activation of translation of pre-existing mRNAs might enable rapid adjustment to environmental changes without the need for de novo transcription. Strikingly, a Puf3p phosphomutant no longer promotes translation but becomes trapped in intracellular foci in an mRNA-dependent manner. Our findings suggest that the inability to properly resolve Puf3p-containing RNA-protein granules via a phosphorylation-based mechanism might be toxic to a cell.

  16. Regulating Transplants

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Legislation to determine brain death is viewed as essential in controlling the organ transplant industry Organ transplant represents a very sensitive and complicated issue. Experts say the temporary administrative regulations recently promulgated by the Central Government are an important step, but relevant laws and regulations must follow. Among these, the

  17. Combination of erlotinib and EGCG induces apoptosis of head and neck cancers through posttranscriptional regulation of Bim and Bcl-2.

    Science.gov (United States)

    Haque, Abedul; Rahman, Mohammad Aminur; Chen, Zhuo Georgia; Saba, Nabil F; Khuri, Fadlo R; Shin, Dong M; Ruhul Amin, A R M

    2015-07-01

    Combinatorial approaches using two or more compounds are gaining increasing attention for cancer therapy. We have previously reported that the combination of the EGFR-TKI erlotinib and epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and by inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. shRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p = 0.005). On the other hand, overexpression of Bcl-2 markedly protected cells from apoptosis (p = 0.003), whereas overexpression of constitutively active AKT only minimally protected cells from apoptosis induced by the combination of the two compounds. Analysis of mRNA expression by RT-PCR revealed that erlotinib, EGCG and their combination had no significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2 suggesting the post-transcriptional regulation of these molecules. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim and stabilized Bim after inhibition of protein translation by cycloheximide. Taken together, our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the posttranscriptional level.

  18. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Xi [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Zhang, Kunshan [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Yanlu; Wang, Junbang; Cui, Yaru [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Li, Siguang, E-mail: siguangli@163.com [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Luo, Yuping, E-mail: luoyuping@163.com [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China)

    2013-10-04

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.

  19. High Temperature Induces Expression of Tobacco Transcription Factor NtMYC2a to Regulate Nicotine and JA Biosynthesis

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    Liming Yang

    2016-10-01

    Full Text Available Environmental stress elevates the level of jasmonic acid (JA and activates the biosynthesis of nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L. by up-regulating the expression of putrescine N-methyltransferase 1 (NtPMT1, which encodes a putrescine N-methyl transferase that catalyzes nicotine formation. The JA signal suppressor JASMONATE ZIM DOMAIN 1 (NtJAZ1 and its target protein, NtMYC2a, also regulate nicotine biosynthesis; however, how these proteins interact to regulate abiotic-induced nicotine biosynthesis is poorly understood. In this study, we found that high-temperature (HT treatment activated transcription of NtMYC2a, which subsequently stimulated the transcription of genes associated with JA biosynthesis, including Lipoxygenase (LOX, Allene oxide synthase (AOS, Allene oxide cyclase (AOC, and 12-oxophytodienodate reductase (OPR. Overexpression of NtMYC2a increased nicotine biosynthesis by enhancing its binding to the promoter of NtPMT1. Overexpression of either NtJAZ1 or proteasome-resistant NtJAZ1∆C suppressed nicotine production under normal conditions, but overexpression only of the former resulted in low levels of nicotine under HT treatment. These data suggest that HT induces NtMYC2a accumulation through increased transcription to activate nicotine synthesis; meanwhile, HT-induced NtMYC2a can activate JA synthesis to promote additional NtMYC2a activity by degrading NtJAZ1 at the post-transcriptional level.

  20. SteatoNet: the first integrated human metabolic model with multi-layered regulation to investigate liver-associated pathologies.

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    Adviti Naik

    2014-12-01

    Full Text Available Current state-of-the-art mathematical models to investigate complex biological processes, in particular liver-associated pathologies, have limited expansiveness, flexibility, representation of integrated regulation and rely on the availability of detailed kinetic data. We generated the SteatoNet, a multi-pathway, multi-tissue model and in silico platform to investigate hepatic metabolism and its associated deregulations. SteatoNet is based on object-oriented modelling, an approach most commonly applied in automotive and process industries, whereby individual objects correspond to functional entities. Objects were compiled to feature two novel hepatic modelling aspects: the interaction of hepatic metabolic pathways with extra-hepatic tissues and the inclusion of transcriptional and post-transcriptional regulation. SteatoNet identification at normalised steady state circumvents the need for constraining kinetic parameters. Validation and identification of flux disturbances that have been proven experimentally in liver patients and animal models highlights the ability of SteatoNet to effectively describe biological behaviour. SteatoNet identifies crucial pathway branches (transport of glucose, lipids and ketone bodies where changes in flux distribution drive the healthy liver towards hepatic steatosis, the primary stage of non-alcoholic fatty liver disease. Cholesterol metabolism and its transcription regulators are highlighted as novel steatosis factors. SteatoNet thus serves as an intuitive in silico platform to identify systemic changes associated with complex hepatic metabolic disorders.

  1. A young Drosophila duplicate gene plays essential roles in spermatogenesis by regulating several Y-linked male fertility genes.

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    Yun Ding

    Full Text Available Gene duplication is supposed to be the major source for genetic innovations. However, how a new duplicate gene acquires functions by integrating into a pathway and results in adaptively important phenotypes has remained largely unknown. Here, we investigated the biological roles and the underlying molecular mechanism of the young kep1 gene family in the Drosophila melanogaster species subgroup to understand the origin and evolution of new genes with new functions. Sequence and expression analysis demonstrates that one of the new duplicates, nsr (novel spermatogenesis regulator, exhibits positive selection signals and novel subcellular localization pattern. Targeted mutagenesis and whole-transcriptome sequencing analysis provide evidence that nsr is required for male reproduction associated with sperm individualization, coiling, and structural integrity of the sperm axoneme via regulation of several Y chromosome fertility genes post-transcriptionally. The absence of nsr-like expression pattern and the presence of the corresponding cis-regulatory elements of the parental gene kep1 in the pre-duplication species Drosophila yakuba indicate that kep1 might not be ancestrally required for male functions and that nsr possibly has experienced the neofunctionalization process, facilitated by changes of trans-regulatory repertories. These findings not only present a comprehensive picture about the evolution of a new duplicate gene but also show that recently originated duplicate genes can acquire multiple biological roles and establish novel functional pathways by regulating essential genes.

  2. Regulation of immunoglobulin gene rearrangement and expression.

    Science.gov (United States)

    Taussig, M J; Sims, M J; Krawinkel, U

    1989-05-01

    The molecular genetic events leading to Ig expression and their control formed the topic of a recent EMBO workshop. This report by Michael Taussig, Martin Sims and Ulrich Krawinkel discusses contributions dealing with genes expressed in early pre-B cells, the mechanism of rearrangement, aberrant rearrangements seen in B cells of SCID mice, the feedback control of rearrangement as studied in transgenic mice, the control of Ig expression at the transcriptional and post-transcriptional levels, and class switching.

  3. miR-8 controls synapse structure by repression of the actin regulator enabled.

    Science.gov (United States)

    Loya, Carlos M; McNeill, Elizabeth M; Bao, Hong; Zhang, Bing; Van Vactor, David

    2014-05-01

    MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play important roles in nervous system development and physiology. However, our understanding of the strategies by which miRNAs control synapse development is limited. We find that the highly conserved miRNA miR-8 regulates the morphology of presynaptic arbors at the Drosophila neuromuscular junction (NMJ) through a postsynaptic mechanism. Developmental analysis shows that miR-8 is required for presynaptic expansion that occurs in response to larval growth of the postsynaptic muscle targets. With an in vivo sensor, we confirm our hypothesis that the founding member of the conserved Ena/VASP (Enabled/Vasodilator Activated Protein) family is regulated by miR-8 through a conserved site in the Ena 3' untranslated region (UTR). Synaptic marker analysis and localization studies suggest that Ena functions within the subsynaptic reticulum (SSR) surrounding presynaptic terminals. Transgenic lines that express forms of a conserved mammalian Ena ortholog further suggest that this localization and function of postsynaptic Ena/VASP family protein is dependent on conserved C-terminal domains known to mediate actin binding and assembly while antagonizing actin-capping proteins. Ultrastructural analysis demonstrates that miR-8 is required for SSR morphogenesis. As predicted by our model, we find that Ena is both sufficient and necessary to account for miR-8-mediated regulation of SSR architecture, consistent with its localization in this compartment. Finally, electrophysiological analysis shows that miR-8 is important for spontaneous neurotransmitter release frequency and quantal content. However, unlike the structural phenotypes, increased expression of Ena fails to mimic the functional defects observed in miR-8-null animals. Together, these findings suggest that miR-8 limits the expansion of presynaptic terminals during larval synapse development through regulation of postsynaptic actin assembly that

  4. Analysis of wide-domain transcriptional regulation in solid-state cultures of Aspergillus oryzae.

    Science.gov (United States)

    McKelvey, Shauna M; Murphy, Richard A

    2010-05-01

    Many filamentous fungi secrete considerable quantities of enzymes including protease, cellulase and xylanase, which are of major industrial importance. Over the past few decades, many of these fungal enzymes have been isolated and their relevant genes characterised. Solid-state fermentation (SSF), an ancient technique described as a fermentation process performed on non-soluble material whereby the material acts as a physical support and as a source of nutrients, is widely employed in the production of industrially important enzymes. Control mechanisms governing gene expression in SSF however, have been rarely studied. The influence of carbon and nitrogen sources on the production and transcriptional regulation of hydrolase enzymes secreted by an Aspergillus strain was investigated with the hope of expanding on the relatively small amount of knowledge regarding cellular control of gene expression. This study involved screening a collection of fungal strains for protease, cellulase and xylanase production under SSF conditions. From this, one fungal strain was then chosen for further analysis. Factors affecting the secretion of the hydrolase enzymes were optimised, and following this, the influence of nutritional supplementation on the production and transcriptional regulation of the enzymes was investigated. Real-time PCR techniques were used to assess the relative expression levels of genes encoding hydrolase activities and of the genes encoding regulatory elements such as AreA, PacC and CreA in an effort to identify possible transcriptional regulation mechanisms. The complexity of gene regulation under SSF conditions became apparent during the study, as other factors such as post-transcriptional regulation appeared to play a far greater role than previously imagined.

  5. Regulation of transcription of cell division genes in the Escherichia coli dcw cluster.

    Science.gov (United States)

    Vicente, M; Gomez, M J; Ayala, J A

    1998-04-01

    The Escherichia coli dcw cluster contains cell division genes, such as the phylogenetically ubiquitous ftsZ, and genes involved in peptidoglycan synthesis. Transcription in the cluster proceeds in the same direction as the progress of the replication fork along the chromosome. Regulation is exerted at the transcriptional and post-transcriptional levels. The absence of transcriptional termination signals may, in principle, allow extension of the transcripts initiated at the up-stream promoter (mraZ1p) even to the furthest down-stream gene (envA). Complementation tests suggest that they extend into ftsW in the central part of the cluster. In addition, the cluster contains other promoters individually regulated by cis- and trans-acting signals. Dissociation of the expression of the ftsZ gene, located after ftsQ and A near the 3' end of the cluster, from its natural regulatory signals leads to an alteration in the physiology of cell division. The complexities observed in the regulation of gene expression in the cluster may then have an important biological role. Among them, LexA-binding SOS boxes have been found at the 5' end of the cluster, preceding promoters which direct the expression of ftsI (coding for PBP3, the penicillin-binding protein involved in septum formation). A gearbox promoter, ftsQ1p, forms part of the signals regulating the transcription of ftsQ, A and Z. It is an inversely growth-dependent mechanism driven by RNA polymerase containing sigma s, the factor involved in the expression of stationary phase-specific genes. Although the dcw cluster is conserved to a different extent in a variety of bacteria, the regulation of gene expression, the presence or absence of individual genes, and even the essentiality of some of them, show variations in the phylogenetic scale which may reflect adaptation to specific life cycles.

  6. Stress-induced nuclear RNA degradation pathways regulate yeast bromodomain factor 2 to promote cell survival.

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    Kevin Roy

    2014-09-01

    Full Text Available Bromodomain proteins are key regulators of gene expression. How the levels of these factors are regulated in specific environmental conditions is unknown. Previous work has established that expression of yeast Bromodomain factor 2 (BDF2 is limited by spliceosome-mediated decay (SMD. Here we show that BDF2 is subject to an additional layer of post-transcriptional control through RNase III-mediated decay (RMD. We found that the yeast RNase III Rnt1p cleaves a stem-loop structure within the BDF2 mRNA to down-regulate its expression. However, these two nuclear RNA degradation pathways play distinct roles in the regulation of BDF2 expression, as we show that the RMD and SMD pathways of the BDF2 mRNA are differentially activated or repressed in specific environmental conditions. RMD is hyper-activated by salt stress and repressed by hydroxyurea-induced DNA damage while SMD is inactivated by salt stress and predominates during DNA damage. Mutations of cis-acting signals that control SMD and RMD rescue numerous growth defects of cells lacking Bdf1p, and show that SMD plays an important role in the DNA damage response. These results demonstrate that specific environmental conditions modulate nuclear RNA degradation pathways to control BDF2 expression and Bdf2p-mediated gene regulation. Moreover, these results show that precise dosage of Bromodomain factors is essential for cell survival in specific environmental conditions, emphasizing their importance for controlling chromatin structure and gene expression in response to environmental stress.

  7. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.

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    Zhong Hua

    Full Text Available MicroRNAs (miRNAs are a class of 20-24 nt non-coding RNAs that regulate gene expression primarily through post-transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle. We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common binding site, or when a functional miRNA competes with a false positive miRNA for the same binding site, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false

  8. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    Science.gov (United States)

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  9. Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation.

    Science.gov (United States)

    Wong, Queenie Wing-Lei; Vaz, Candida; Lee, Qian Yi; Zhao, Tian Yun; Luo, Raymond; Archer, Stuart K; Preiss, Thomas; Tanavde, Vivek; Vardy, Leah A

    2016-01-01

    The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

  10. Trans-Regulation of RNA-Binding Protein Motifs by MicroRNA

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    Scott eTenenbaum

    2014-04-01

    Full Text Available The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.

  11. Function and Regulation of MicroRNAs and Their Potential as Biomarkers in Paediatric Liver Disease

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    Diego A. Calvopina

    2016-10-01

    Full Text Available MicroRNAs (miRNAs are short non-coding RNAs involved in biological and pathological processes of every cell type, including liver cells. Transcribed from specific genes, miRNA precursors are processed in the cytoplasm into mature miRNAs and as part of the RNA-induced silencing complex (RISC complex binds to messenger RNA (mRNA by imperfect complementarity. This leads to the regulation of gene expression at a post-transcriptional level. The function of a number of different miRNAs in fibrogenesis associated with the progression of chronic liver disease has recently been elucidated. Furthermore, miRNAs have been shown to be both disease-and tissue-specific and are stable in the circulation, which has led to increasing investigation on their utility as biomarkers for the diagnosis of chronic liver diseases, including those in children. Here, we review the current knowledge on the biogenesis of microRNA, the mechanisms of translational repression and the use of miRNA as circulatory biomarkers in chronic paediatric liver diseases including cystic fibrosis associated liver disease, biliary atresia and viral hepatitis B.

  12. MeCP2: multifaceted roles in gene regulation and neural development.

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    Cheng, Tian-Lin; Qiu, Zilong

    2014-08-01

    Methyl-CpG-binding protein 2 (MeCP2) is a classic methylated-DNA-binding protein, dysfunctions of which lead to various neurodevelopmental disorders such as Rett syndrome and autism spectrum disorder. Initially recognized as a transcriptional repressor, MeCP2 has been studied extensively and its functions have been expanded dramatically in the past two decades. Recently, it was found to be involved in gene regulation at the post-transcriptional level. MeCP2 represses nuclear microRNA processing by interacting directly with the Drosha/DGCR8 complex. In addition to its multifaceted functions, MeCP2 is remarkably modulated by posttranslational modifications such as phosphorylation, SUMOylation, and acetylation, providing more regulatory dimensions to its functions. The role of MeCP2 in the central nervous system has been studied extensively, from neurons to glia. Future investigations combining molecular, cellular, and physiological methods are necessary for defining the roles of MeCP2 in the brain and developing efficient treatments for MeCP2-related brain disorders.

  13. HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

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    Jing Jin

    Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

  14. Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

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    David J Leslie

    2015-07-01

    Full Text Available Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

  15. Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation.

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    Queenie Wing-Lei Wong

    Full Text Available The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs and in ESC derived Neural Precursor Cells (NPCs using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.

  16. A perspective on the role of microRNA-128 regulation in mental and behavioral disorders

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    Ai Sze eChing

    2015-12-01

    Full Text Available MiRNAs are short, non-coding RNA molecules that regulate gene expression post-transcriptionally. Over the past decade, misregulated miRNA pathways have been associated with various diseases such as cancer, neurodegenerative diseases and neurodevelopmental disorders. In this article, we aim to discuss the role played by miR-128 in neuropsychiatric disorders, and highlight potential target genes from an in silico analysis of predicted miR-128 targets. We also discuss the differences of target gene determination based on a bioinformatics or empirical approach. Using data from TargetScan and published reports, we narrowed the miR-128 target gene list to those that are known to be associated with neuropsychiatric disorders, and found that these genes can be classified into 29 gene clusters and are mostly enriched in cancer and MAPK signaling pathways. We also highlight some recent studies on several of the miR-128 targets which should be investigated further as potential candidate genes for therapeutic interventions.

  17. The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

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    Alexandra Steffens

    2015-07-01

    Full Text Available Members of the highly conserved class of BEACH domain containing proteins (BDCPs have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1, associates to mRNA processing bodies (P-bodies, and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

  18. Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

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    Knight, John R P; Bastide, Amandine; Roobol, Anne; Roobol, Jo; Jackson, Thomas J; Utami, Wahyu; Barrett, David A; Smales, C Mark; Willis, Anne E

    2015-01-15

    Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

  19. MicroRNA-regulation of Anopheles gambiae immunity to Plasmodium falciparum infection and midgut microbiota.

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    Dennison, Nathan J; BenMarzouk-Hidalgo, Omar J; Dimopoulos, George

    2015-03-01

    Invasion of the malaria vector Anopheles gambiae midgut by Plasmodium parasites triggers transcriptional changes of immune genes that mediate the antiparasitic defense. This response is largely regulated by the Toll and Immune deficiency (IMD) pathways. To determine whether A. gambiae microRNAs (miRNAs) are involved in regulating the anti-Plasmodium defense, we showed that suppression of miRNA biogenesis results in increased resistance to Plasmodium falciparum infection. In silico analysis of A. gambiae immune effector genes identified multiple transcripts with miRNA binding sites. A comparative miRNA microarray abundance analysis of P. falciparum infected and naïve mosquito midgut tissues showed elevated abundance of miRNAs aga-miR-989 and aga-miR-305 in infected midguts. Antagomir inhibition of aga-miR-305 increased resistance to P. falciparum infection and suppressed the midgut microbiota. Conversely, treatment of mosquitoes with an artificial aga-miR-305 mimic increased susceptibility to P. falciparum infection and resulted in expansion of midgut microbiota, suggesting that aga-miR-305 acts as a P. falciparum and gut microbiota agonist by negatively regulating the mosquito immune response. In silico prediction of aga-miR-305 target genes identified several anti-Plasmodium effectors. Our study shows that A. gambiae aga-miR-305 regulates the anti-Plasmodium response and midgut microbiota, likely through post-transcriptional modification of immune effector genes.

  20. Sex-different and growth hormone-regulated expression of microRNA in rat liver

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    Tollet-Egnell Petra

    2009-02-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are short non-coding RNAs playing an important role in post-transcriptional regulation of gene expression. We have previously shown that hepatic transcript profiles are different between males and females; that some of these differences are under the regulation of growth hormone (GH; and that mild starvation diminishes some of the differences. In this study, we tested if hepatic miRNAs are regulated in a similar manner. Results Using microarrays, miRNA screening was performed to identify sex-dependent miRNAs in rat liver. Out of 324 unique probes on the array, 254 were expressed in the liver and eight (3% of 254 of those were found to be different between the sexes. Among the eight putative sex-different miRNAs, only one female-predominant miRNA (miR-29b was confirmed using quantitative real-time PCR. Furthermore, 1 week of continuous GH-treatment in male rats reduced the levels of miR-451 and miR-29b, whereas mild starvation (12 hours raised the levels of miR-451, miR-122a and miR-29b in both sexes. The biggest effects were obtained on miR-29b with GH-treatment. Conclusion We conclude that hepatic miRNA levels depend on the hormonal and nutritional status of the animal and show that miR-29b is a female-predominant and GH-regulated miRNA in rat liver.

  1. Inferring yeast cell cycle regulators and interactions using transcription factor activities

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    Galbraith Simon J

    2005-06-01

    Full Text Available Abstract Background Since transcription factors are often regulated at the post-transcriptional level, their activities, rather than expression levels may provide valuable information for investigating functions and their interactions. The recently developed Network Component Analysis (NCA and its generalized form (gNCA provide a robust framework for deducing the transcription factor activities (TFAs from various types of DNA microarray data and transcription factor-gene connectivity. The goal of this work is to demonstrate the utility of TFAs in inferring transcription factor functions and interactions in Saccharomyces cerevisiae cell cycle regulation. Results Using gNCA, we determined 74 TFAs from both wild type and fkh1 fkh2 deletion mutant microarray data encompassing 1529 ORFs. We hypothesized that transcription factors participating in the cell cycle regulation exhibit cyclic activity profiles. This hypothesis was supported by the TFA profiles of known cell cycle factors and was used as a basis to uncover other potential cell cycle factors. By combining the results from both cluster analysis and periodicity analysis, we recovered nearly 90% of the known cell cycle regulators, and identified 5 putative cell cycle-related transcription factors (Dal81, Hap2, Hir2, Mss11, and Rlm1. In addition, by analyzing expression data from transcription factor knockout strains, we determined 3 verified (Ace2, Ndd1, and Swi5 and 4 putative interaction partners (Cha4, Hap2, Fhl1, and Rts2 of the forkhead transcription factors. Sensitivity of TFAs to connectivity errors was determined to provide confidence level of these predictions. Conclusion By subjecting TFA profiles to analyses based upon physiological signatures we were able to identify cell cycle related transcription factors consistent with current literature, transcription factors with potential cell cycle dependent roles, and interactions between transcription factors.

  2. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.

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    Bretschneider, Maria; Busch, Bianca; Mueller, Daniel; Nolze, Alexander; Schreier, Barbara; Gekle, Michael; Grossmann, Claudia

    2016-04-01

    Inappropriately activated mineralocorticoid receptor (MR) is a risk factor for vascular remodeling with unclear molecular mechanism. Recent findings suggest that post-transcriptional regulation by micro-RNAs (miRs) may be involved. Our aim was to search for MR-dependent miRs in vascular smooth muscle cells (VSMCs) and to explore the underlying molecular mechanism and the pathologic relevance. We detected that aldosteroneviathe MR reduces miR-29bin vivoin murine aorta and in human primary and cultured VSMCs (ED50= 0.07 nM) but not in endothelial cells [quantitative PCR (qPCR), luciferase assays]. This effect was mediated by an increased decay of miR-29b in the cytoplasm with unchanged miR-29 family member or primary-miR levels. Decreased miR-29b led to an increase in extracellular matrix measured by ELISA and qPCR and enhanced VSMC migration in single cell-tracking experiments. Additionally, cell proliferation and the apoptosis/necrosis ratio (caspase/lactate dehydrogenase assay) was modulated by miR-29b. Enhanced VSMC migration by aldosterone required miR-29b regulation. Control experiments were performed with scrambled RNA and empty plasmids, by comparing aldosterone-stimulated with vehicle-incubated cells. Overall, our findings provide novel insights into the molecular mechanism of aldosterone-mediated vascular pathogenesis by identifying miR-29b as a pathophysiologic relevant target of activated MR in VSMCs and by highlighting the importance of miR processing for miR regulation.-Bretschneider, M., Busch, B., Mueller, D., Nolze, A., Schreier, B., Gekle, M., Grossmann, C. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.

  3. Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.

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    Noam Leviatan

    Full Text Available Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.

  4. Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.

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    Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

    2013-01-01

    Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.

  5. MicroRNA调控昆虫免疫的研究进展%Advances in research on the role of microRNAs in the regulation of insect immunity

    Institute of Scientific and Technical Information of China (English)

    徐燕霞; 刘晓君; 唐斌; 王世贵

    2015-01-01

    MicroRNA ( miRNA) are a class of endogenous genes encoding a length of about 21 nt non-coding single-stranded RNA molecules that play an important role in the regulation of gene expression at the post-transcriptional level in various biological processes. This paper summarizes progress in research on the role of miRNAs in the regulation of insect immune defense, including their role in regulating gene expression of insect antimicrobial peptides, regulation of insect antiviral immune response, regulating the immune response of insect and endosymbiotic bacteria, and regulation the insect immune response to pathogens, etc.%MicroRNA(miRNA)是一类由内源基因编码的长度约为21 nt的非编码单链RNA分子,在各种生物学过程中具有转录后水平调控基因表达的重要作用。本文从miRNA调控昆虫抗菌肽的表达、调控昆虫抗病毒免疫反应、调控昆虫与共生菌的免疫反应及调控昆虫作为病原体媒介引起的免疫反应等方面,就miRNAs在昆虫免疫防御方面的研究进展作一综述。

  6. MicroRNA-155 Regulates Inflammatory Cytokine Production in Tumor-associated Macrophages via Targeting C/EBPβ

    Institute of Scientific and Technical Information of China (English)

    Min He; Zhenqun Xu; Tong Ding; Dong-Ming Kuang; Limin Zheng

    2009-01-01

    Macrophages (Mψ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mψ to trigger transient activation of monocytes, but the underlying regulatory mechanisms are incompletely understood. Here, we showed that the protein expression of transcription factor C/EBPβ was markedly elevated in tumor-associated Mψ both in vitro and human tumors in situ. The expression of C/EBPβ protein correlated with cytokine production in tumor-activated monocytes. Moreover, we found that C/EBPβ expression was regulated at the post-transcriptional level and correlated with sustained reduction of microRNA-155 (miR-155) in tumor-activated monocytes. Bioinformatic analysis revealed that C/EBPβ is a potential target of miR-155 and luciferase assay confirmed that C/EBPβ translation is suppressed by miR-155 through interaction with the 3'UTR of C/EBPβ mRNA. Further analysis showed that induction of miR-155 suppressed C/EBPβ protein expression as well as cytokine production in tumor-activated monocytes, an effect which could be mimicked by silencing of C/EBPβ. These results indicate that tumor environment causes a sustained reduction of miR-155 in monocytes/Mψ, which in turn regulates the functional activities of monocytes/Mψ by releasing the translational inhibition of transcription factor C/EBPβ. Cellular & Molecular Immunology. 2009;6(5):343-352.

  7. miR-297 modulates multidrug resistance in human colorectal carcinoma by down-regulating MRP-2.

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    Xu, Ke; Liang, Xin; Shen, Ke; Cui, Daling; Zheng, Yuanhong; Xu, Jianhua; Fan, Zhongze; Qiu, Yanyan; Li, Qi; Ni, Lei; Liu, Jianwen

    2012-09-01

    Colorectal carcinoma is a frequent cause of cancer-related death in men and women. miRNAs (microRNAs) are endogenous small non-coding RNAs that regulate gene expression negatively at the post-transcriptional level. In the present study we investigated the possible role of microRNAs in the development of MDR (multidrug resistance) in colorectal carcinoma cells. We analysed miRNA expression levels between MDR colorectal carcinoma cell line HCT116/L-OHP cells and their parent cell line HCT116 using a miRNA microarray. miR-297 showed lower expression in HCT116/L-OHP cells compared with its parental cells. MRP-2 (MDR-associated protein 2) is an important MDR protein in platinum-drug-resistance cells and is a predicted target of miR-297. Additionally miR-297 was down-regulated in a panel of human colorectal carcinoma tissues and negatively correlated with expression levels of MRP-2. Furthermore, we found that ectopic expression of miR-297 in MDR colorectal carcinoma cells reduced MRP-2 protein level and sensitized these cells to anti-cancer drugs in vitro and in vivo. Taken together, our findings suggest that miR-297 could play a role in the development of MDR in colorectal carcinoma cells, at least in part by modulation of MRP-2.

  8. Insights into the regulation of human CNV-miRNAs from the view of their target genes

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    Wu Xudong

    2012-12-01

    Full Text Available Abstract Background microRNAs (miRNAs represent a class of small (typically 22 nucleotides in length non-coding RNAs that can degrade their target mRNAs or block their translation. Recent research showed that copy number alterations of miRNAs and their target genes are highly prevalent in cancers; however, the evolutionary and biological functions of naturally existing copy number variable miRNAs (CNV-miRNAs among individuals have not been studied extensively throughout the genome. Results In this study, we comprehensively analyzed the properties of genes regulated by CNV-miRNAs, and found that CNV-miRNAs tend to target a higher average number of genes and prefer to synergistically regulate the same genes; further, the targets of CNV-miRNAs tend to have higher variability of expression within and between populations. Finally, we found the targets of CNV-miRNAs are more likely to be differentially expressed among tissues and developmental stages, and participate in a wide range of cellular responses. Conclusions Our analyses of CNV-miRNAs provide new insights into the impact of copy number variations on miRNA-mediated post-transcriptional networks. The deeper interpretation of patterns of gene expression variation and the functional characterization of CNV-miRNAs will help to broaden the current understanding of the molecular basis of human phenotypic diversity.

  9. Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

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    Jennifer Koenig

    Full Text Available The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A and human neuroblastoma (SH-SY5Y cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

  10. Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia.

    Science.gov (United States)

    Koenig, Jennifer; Werdehausen, Robert; Linley, John E; Habib, Abdella M; Vernon, Jeffrey; Lolignier, Stephane; Eijkelkamp, Niels; Zhao, Jing; Okorokov, Andrei L; Woods, C Geoffrey; Wood, John N; Cox, James J

    2015-01-01

    The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

  11. Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation

    Science.gov (United States)

    Weston, Andrew; Sommerville, John

    2006-01-01

    The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2α, bind to nascent transcripts on chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression, mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation, storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages of their lifetimes marks them out as key regulators of post-transcriptional gene expression. PMID:16769775

  12. Jeb/Alk signalling regulates the Lame duck GLI family transcription factor in the Drosophila visceral mesoderm.

    Science.gov (United States)

    Popichenko, Dmitry; Hugosson, Fredrik; Sjögren, Camilla; Dogru, Murat; Yamazaki, Yasuo; Wolfstetter, Georg; Schönherr, Christina; Fallah, Mahsa; Hallberg, Bengt; Nguyen, Hanh; Palmer, Ruth H

    2013-08-01

    The Jelly belly (Jeb)/Anaplastic Lymphoma Kinase (Alk) signalling pathway regulates myoblast fusion in the circular visceral mesoderm (VM) of Drosophila embryos via specification of founder cells. However, only a limited number of target molecules for this pathway are described. We have investigated the role of the Lame Duck (Lmd) transcription factor in VM development in relationship to Jeb/Alk signal transduction. We show that Alk signalling negatively regulates Lmd activity post-transcriptionally through the MEK/MAPK (ERK) cascade resulting in a relocalisation of Lmd protein from the nucleus to cytoplasm. It has previously been shown that downregulation of Lmd protein is necessary for the correct specification of founder cells. In the visceral mesoderm of lmd mutant embryos, fusion-competent myoblasts seem to be converted to 'founder-like' cells that are still able to build a gut musculature even in the absence of fusion. The ability of Alk signalling to downregulate Lmd protein requires the N-terminal 140 amino acids, as a Lmd(141-866) mutant remains nuclear in the presence of active ALK and is able to drive robust expression of the Lmd downstream target Vrp1 in the developing VM. Our results suggest that Lmd is a target of Jeb/Alk signalling in the VM of Drosophila embryos.

  13. The Nicastrin-like protein Nicalin regulates assembly and stability of the Nicalin-nodal modulator (NOMO) membrane protein complex.

    Science.gov (United States)

    Haffner, Christof; Dettmer, Ulf; Weiler, Timotheus; Haass, Christian

    2007-04-06

    The assembly of the gamma-secretase complex, an Alzheimer disease-related protease required for beta-amyloid generation, is tightly regulated and predominantly limited by the stoichiometrical availability of its components. We have identified a novel endoplasmic reticulum-located protein complex that is regulated in a similar fashion. It contains the recently identified Nodal signaling antagonists Nicalin (a distant homolog of the gamma-secretase component Nicastrin) and NOMO (Nodal modulator). Using an RNA interference approach, we found that Nicalin and NOMO became unstable in the absence of the respective binding partner, suggesting that complex formation has a stabilizing effect. Overexpression of Nicalin resulted in an increase in NOMO, whereas endogenous Nicalin was reduced below the detection limit. Both effects were shown to occur at a post-transcriptional level. Thus, NOMO is most likely produced in excess amounts and either stabilized by Nicalin or rapidly degraded. In contrast, Nicalin levels are limited independently of NOMO. We, therefore, propose that Nicalin controls the assembly and stability of the Nicalin-NOMO complex.

  14. Down-regulation of miR-181a can reduce heat stress damage in PBMCs of Holstein cows.

    Science.gov (United States)

    Chen, Kun-Lin; Fu, Yuan-Yuan; Shi, Min-Yan; Li, Hui-Xia

    2016-09-01

    Heat stress can weaken the immune system and even increase livestock's susceptibility to disease. MicroRNA (miR) is short non-coding RNA that functions in post-transcriptional regulation of gene expression and some phenotypes. Our recent study found that miR-181a is highly expressed in the serum of heat-stressed Holstein cows, but the potential function of miR-181a is still not clarified. In this study, peripheral blood mononuclear cells (PBMCs), isolated from Holstein cows' peripheral blood, were used to investigate the effects of miR-181a inhibitor on heat stress damage. Our results showed that significant apoptosis and oxidative damage were induced by heat stress in PBMCs. However, with apoptosis, the levels of reactive oxygen species (ROS) and content of malondialdehyde (MDA) were reduced, while the content of glutathione (GSH) and the activity of superoxide dismutase (SOD) were increased even under heat stress conditions after transfecting miR-181a inhibitors to PBMCs. Meanwhile, mRNA expression of bax and caspase-3 was significantly decreased, but mRNA expression of bcl-2 was increased in transfected PBMCs. In conclusion, our results demonstrated that down-regulation of miR-181a can reduce heat stress damage in PBMCs of Holstein cows.

  15. Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.

    Science.gov (United States)

    Bilichak, Andriy; Yao, Youli; Kovalchuk, Igor

    2014-06-01

    Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue.

  16. MicroRNA-dependent regulation of telomere maintenance mechanisms: a field as much unexplored as potentially promising.

    Science.gov (United States)

    Santambrogio, Francesca; Gandellini, Paolo; Cimino-Reale, Graziella; Zaffaroni, Nadia; Folini, Marco

    2014-01-01

    The activation of telomere maintenance mechanisms, which rely on telomerase reactivation or on a recombination-based process known as alternative lengthening of telomeres, guarantees a limitless proliferative potential to human tumor cells. To date, the molecular underpinnings that drive the activation of telomere maintenance mechanisms during tumorigenesis are poorly understood, but there are indications that complex signaling networks might be involved. Since telomerase activity has been mainly detected in tumors of epithelial origin and the alternative lengthening of telomere mechanisms is more frequently expressed in mesenchymal and neuroepithelial cancers, it could be hypothesized that cell-type specific mechanisms can favor their activation during tumor development. In this context, microRNAs - small non coding RNAs that regulate gene expression at post-transcriptional level - have emerged as key players in the development and progression of human cancers, being involved in the control of all the typical features of cancer cells, including the limitless replicative potential. In the present review, we will summarize the recent findings concerning the identification and biological validation of microRNAs which may play a role in the regulation of telomere biology as well as of the mechanisms that govern telomere maintenance.

  17. MicroRNA-181b regulates endotoxin tolerance by targeting IL-6 in macrophage RAW264.7 cells.

    Science.gov (United States)

    Zhang, Wenjun; Shen, Xiaojun; Xie, Luyang; Chu, Maoping; Ma, Yanmei

    2015-01-01

    Interleukin 6 (IL-6) is a major pro-inflammatory cytokine and dysregulation of IL-6 is relevant to many inflammatory diseases. Endotoxin induced tolerance of IL-6 is an important mechanism to avoid the excessive immune reaction. But to date, the molecular mechanisms of endotoxin tolerance of IL-6 remain unclear. Here we reported that IL-6 secretion and microRNA-181b (miR-181b) expression were inversely correlated following LPS stimulation. We also demonstrated that miR-181b targeting the 3'-UTR of IL-6 transcripts and up-regulation of miR-181b was associated with NF-kB. We further demonstrated that up-regulation of miR-181b in response to LPS was required for inducing IL-6 tolerance in macrophage. Our results suggested that the post-transcriptional control mediated by miR-181b could be involved in fine tuning the critical level of IL-6 expression in endotoxin tolerance.

  18. NORM regulations

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P. [ed.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  19. Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Carmen Espinoza

    Full Text Available In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further

  20. Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells.

    Science.gov (United States)

    Zhong, Xiaomin; Li, Ning; Liang, Shun; Huang, Qihong; Coukos, George; Zhang, Lin

    2010-12-31

    LIN28 (a homologue of the Caenorhabditis elegans lin-28 gene) is an evolutionarily conserved RNA-binding protein and a master regulator controlling the pluripotency of embryonic stem cells. Together with OCT4, SOX2, and NANOG, LIN28 can reprogram somatic cells, producing induced pluripotent stem cells. Expression of LIN28 is highly restricted to embryonic stem cells and developing tissues. In human tumors, LIN28 is up-regulated and functions as an oncogene promoting malignant transformation and tumor progression. However, the mechanisms of transcriptional and post-transcriptional regulation of LIN28 are still largely unknown. To examine microRNAs (miRNAs) that repress LIN28 expression, a combined in silico prediction and miRNA library screening approach was used in the present study. Four miRNAs directly regulating LIN28 (let-7, mir-125, mir-9, and mir-30) were initially identified by this approach and further validated by quantitative RT-PCR, Western blot analysis, and a LIN28 3'-UTR reporter assay. We found that expression levels of these four miRNAs were clustered together and inversely correlated with LIN28 expression during embryonic stem cell differentiation. In addition, the expression of these miRNAs was remarkably lower in LIN28-positive tumor cells compared with LIN28-negative tumor cells. Importantly, we demonstrated that these miRNAs were able to regulate the expression and activity of let-7, mediated by LIN28. Taken together, our studies demonstrate that miRNAs let-7, mir-125, mir-9, and mir-30 directly repress LIN28 expression in embryonic stem and cancer cells. Global down-regulation of these miRNAs may be one of the mechanisms of LIN28 reactivation in human cancers.

  1. MiR-138 indirectly regulates the MDR1 promoter by NF-κB/p65 silencing.

    Science.gov (United States)

    Requenez-Contreras, J L; López-Castillejos, E S; Hernández-Flores, R; Moreno-Eutimio, M A; Granados-Riveron, J T; Martinez-Ruiz, G U; Aquino-Jarquin, G

    2017-03-11

    MicroRNAs (miRNAs) are known to mediate post-transcriptional gene silencing in the cytoplasm and recent evidence indicates that may also possess nuclear roles in regulating gene expression. A previous study showed that miR-138 is involved in the multidrug resistance of leukemia cells through down-regulation of the drug efflux pump P-glycoprotein (P-gp), the protein encoded by the human multidrug-resistant ABCB1/MDR1 gene. However, the transcriptional regulatory mechanisms responsible remain to be elucidated. To deepen the description of the mechanism of transcriptional gene silencing on the MDR1 promoter, we initially performed a bioinformatics search for potential miR-138 binding sites in the MDR1 gene promoter sequence. Interestingly, we did not find miR-138 binding sites in this region, suggesting an indirect regulation. From six representative transcriptional factors involved in MDR1 gene regulation, an in silico analysis revealed that NF-κB/p65 has a specific binding site for miR-138. The results of luciferase reporter assay, western blot and flow cytometry shown here suggest that miR-138 might modulate the human MDR1 expression by inhibiting NF-κB/p65 as an indirect mechanism of MDR1 regulation. Furthermore, employing the human macrophage-like cell line U937 we observed comparable results with NF-κB/p65 down-regulation and we also observed a significant reduction in the IL-6 and TNF-α mRNA, as well as in their secreted pro-inflammatory cytokines following miR-138 expression, suggesting that canonical NF-κB target genes might also be potential targets for miR-138 in leukemia cells.

  2. Small RNAs regulate the biocontrol property of fluorescent Pseudomonas strain Psd.

    Science.gov (United States)

    Upadhyay, Anamika; Kochar, Mandira; Upadhyay, Ashutosh; Tripathy, Soumya; Rajam, Manchikatla Venkat; Srivastava, Sheela

    2017-03-01

    The production of biocontrol factors by Pseudomonads is reported to be controlled at the post-transcriptional level by the GacS/GacA signal transduction pathway. This involves RNA-binding translational repressor proteins, RsmA and RsmE, and the small regulatory RNAs (sRNAs) RsmX, RsmY, and RsmZ. While the former represses genes involved in secondary metabolite production, the latter relieves this repression at the end of exponential growth. We have studied the fluorescent Pseudomonas strain Psd, possessing good biocontrol potential, and confirmed the presence of rsmY and rsmZ by PCR amplification. Gene constructs for all the three small RNAs (RsmX, RsmY and RsmZ) carried on broad host-range plasmid, pME6032 were mobilized into strain Psd. Expression analysis of gacA in the recombinant strains over-expressing rsmX (Psd-pME7320), rsmY (Psd-pME6359) and rsmZ (Psd-pME6918) revealed a significant upregulation of the response regulator. Besides, a remarkable down-regulation of rsmA was also reported in all the strains. The variant strains were found to produce comparatively higher levels of phenazines. Indole acetic acid levels were higher to some extent, and strain Psd-pME6918 also showed elevated production of HCN. The tomato seedlings infected with Fusarium oxysporum and Verticillium dahliae in the presence of culture filtrate of the recombinant strains showed better plant protection response in comparison to the wild-type strain Psd. These results suggest that small RNAs are important determinants in regulation of the biocontrol property of strain Psd.

  3. Regulation of the expression of nitric oxide synthase by Leishmania mexicana amastigotes in murine dendritic cells.

    Science.gov (United States)

    Wilkins-Rodríguez, Arturo A; Escalona-Montaño, Alma Reyna; Aguirre-García, Magdalena; Becker, Ingeborg; Gutiérrez-Kobeh, Laila

    2010-11-01

    In mammalian hosts, Leishmania parasites are obligatory intracellular organisms that invade macrophages (M phi) and dendritic cells (DC). In M phi, the production of nitric oxide (NO) catalyzed by the inducible nitric oxide synthase (iNOS) has been implicated as a major defense against Leishmania infection. The modulation of this microbicidal mechanism by different species of Leishmania has been well studied in M phi. Although DC are permissive for infection with Leishmania both in vivo and in vitro, the effect of this parasite in the expression of iNOS and NO production in these cells has not been established. To address this issue, we analyzed the regulation of iNOS by Leishmania mexicana amastigotes in murine bone marrow-derived dendritic cells (BMDC) stimulated with LPS and IFN-gamma. We show that the infection of BMDC with amastigotes down regulated NO production and diminished iNOS protein levels in cells stimulated with LPS alone or in combination with IFN-gamma. The reduction in iNOS protein levels and NO production did not correlate with a decrease in iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Although amastigotes were able to reduce NO production in BMDC, the interference with this cytotoxic mechanism was not sufficient to permit the survival of L. mexicana. At 48 h post-infection, BMDC stimulated with LPS+IFN-gamma were able to eliminate the parasites. These results are the first to identify the regulation of iNOS by L. mexicana amastigotes in DC.

  4. Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

    Energy Technology Data Exchange (ETDEWEB)

    Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki; Yagi, Hiroaki; Shigaki-Miyamoto, Kaya; Toyota, Syukichi; Azuma, Yuko [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Chuo-ku, Kumamoto 860-8555 (Japan); Igarashi, Masayuki [Laboratory of Disease Biology, Institute of Microbial Chemistry, Shinagawa-ku, Tokyo 141-0021 (Japan); Tani, Tokio, E-mail: ttani@sci.kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Chuo-ku, Kumamoto 860-8555 (Japan)

    2014-03-28

    Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

  5. MiR-125a-3p timely inhibits oligodendroglial maturation and is pathologically up-regulated in human multiple sclerosis

    Science.gov (United States)

    Lecca, Davide; Marangon, Davide; Coppolino, Giusy T.; Méndez, Aida Menéndez; Finardi, Annamaria; Costa, Gloria Dalla; Martinelli, Vittorio; Furlan, Roberto; Abbracchio, Maria P.

    2016-01-01

    In the mature central nervous system (CNS), oligodendrocytes provide support and insulation to axons thanks to the production of a myelin sheath. During their maturation to myelinating cells, oligodendroglial precursors (OPCs) follow a very precise differentiation program, which is finely orchestrated by transcription factors, epigenetic factors and microRNAs (miRNAs), a class of small non-coding RNAs involved in post-transcriptional regulation. Any alterations in this program can potentially contribute to dysregulated myelination, impaired remyelination and neurodegenerative conditions, as it happens in multiple sclerosis (MS). Here, we identify miR-125a-3p, a developmentally regulated miRNA, as a new actor of oligodendroglial maturation, that, in the mammalian CNS regulates the expression of myelin genes by simultaneously acting on several of its already validated targets. In cultured OPCs, over-expression of miR-125a-3p by mimic treatment impairs while its inhibition with an antago-miR stimulates oligodendroglial maturation. Moreover, we show that miR-125a-3p levels are abnormally high in the cerebrospinal fluid of MS patients bearing active demyelinating lesions, suggesting that its pathological upregulation may contribute to MS development, at least in part by blockade of OPC differentiation leading to impaired repair of demyelinated lesions. PMID:27698367

  6. The 3' untranslated region of the two cytosolic glutamine synthetase (GS(1)) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine.

    Science.gov (United States)

    Simon, Bindu; Sengupta-Gopalan, Champa

    2010-10-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to produce glutamine. The GS enzyme is located either in the chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene family and the members exhibit differential expression pattern mostly attributed to transcriptional regulation. Based on our recent finding that a soybean GS(1) gene, Gmglnβ ( 1 ) is subject to its 3'UTR-mediated post-transcriptional regulation as a transgene in alfalfa (Medicago sativa) we have raised the question of whether the 3'UTR-mediated transcript destabilization is a more universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes, MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these transformants suggests that while both the 3'UTRs promote transcript turnover, the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs along with Gmglnβ ( 1 ) 3'UTR respond to nitrate as a trigger in transcript turnover. More detailed analysis points to glutamine rather than nitrate as the mediator of transcript turnover. Our data suggests that the 3'UTR-mediated regulation of GS(1) genes at the level of transcript turnover is probably universal and is used for fine-tuning the expression in keeping with the availability of the substrates.

  7. Genome-wide identification of small RNAs in Bifidobacterium animalis subsp. lactis KLDS 2.0603 and their regulation role in the adaption to gastrointestinal environment.

    Directory of Open Access Journals (Sweden)

    De-Quan Zhu

    Full Text Available Bifidobacteria are one of the predominant bacterial species in the human gastrointestinal tract (GIT and play a vital role in the host's health by acting as probiotics. However, how they regulate themselves to adapt to GIT of their host remains unknown.Eighteen bifidobacterial strains were used to analyze their adaptive capacities towards simulated GIT environment. The strain with highest survival rate and adhesion ability was selected for comparative genome as well as transcriptomic analysis.The Bifidobacterium animalis subsp. lactis KLDS 2.0603 strain was demonstrated to have the highest survival rate and adhesion ability in simulated GIT treatments. The comparative genome analysis revealed that the KLDS 2.0603 has most similar whole genome sequence compared with BB-12 strain. Eleven intergenic sRNAs were identified after genomes prediction and transcriptomic analysis of KLDS 2.0603. Transcriptomic analysis also showed that genes (mainly sRNAs targeted genes and sRNAs were differentially expressed in different stress conditions, suggesting that sRNAs might play a crucial role in regulating genes involved in the stress resistance of this strain towards environmental changes.This study first provided deep and comprehensive insights into the regulation of KLDS 2.0603 strain at transcription and post-transcription level towards environmental.

  8. Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress.

    Directory of Open Access Journals (Sweden)

    Stacey L Lehman

    2015-06-01

    Full Text Available Multiple transcripts encode for the cell cycle inhibitor p21(Cip1. These transcripts produce identical proteins but differ in their 5' untranslated regions (UTRs. Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through (35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5' upstream open reading frames (uORFs through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

  9. MiR-21 is an Ngf-modulated microRNA that supports Ngf signaling and regulates neuronal degeneration in PC12 cells.

    Science.gov (United States)

    Montalban, Enrica; Mattugini, Nicola; Ciarapica, Roberta; Provenzano, Claudia; Savino, Mauro; Scagnoli, Fiorella; Prosperini, Gianluca; Carissimi, Claudia; Fulci, Valerio; Matrone, Carmela; Calissano, Pietro; Nasi, Sergio

    2014-06-01

    The neurotrophins Ngf, Bdnf, NT-3, NT4-5 have key roles in development, survival, and plasticity of neuronal cells. Their action involves broad gene expression changes at the level of transcription and translation. MicroRNAs (miRs)-small RNA molecules that control gene expression post-transcriptionally-are increasingly implicated in regulating development and plasticity of neural cells. Using PC12 cells as a model system, we show that Ngf modulates changes in expression of a variety of microRNAs, including miRs known to be modulated by neurotrophins-such as the miR-212/132 cluster-and several others, such as miR-21, miR-29c, miR-30c, miR-93, miR-103, miR-207, miR-691, and miR-709. Pathway analysis indicates that Ngf-modulated miRs may regulate many protein components of signaling pathways involved in neuronal development and disease. In particular, we show that miR-21 enhances neurotrophin signaling and controls neuronal differentiation induced by Ngf. Notably, in a situation mimicking neurodegeneration-differentiated neurons deprived of Ngf-this microRNA is able to preserve the neurite network and to support viability of the neurons. These findings uncover a broad role of microRNAs in regulating neurotrophin signaling and suggest that aberrant expression of one or more Ngf-modulated miRs may be involved in neurodegenerative diseases.

  10. MiR-128b is down-regulated in gastric cancer and negatively regulates tumour cell viability by targeting PDK1/Akt/NF-κB axis

    Indian Academy of Sciences (India)

    Ling Zhang; Jun Lei; Zi-Ling Fang; Jian-Ping Xiong

    2016-03-01

    Gastric cancer (GC) is the fourth most prevalent type of cancer worldwide, which is usually caused by the interaction between environmental and genetic factors, or epigenetic aspects. Referring to the non-coding RNAs, miR-128b has been reported to be associated with many tumour cases, and exerts distinct functions in different types of cancers. However, the function of miR-128b in GC onset and progression largely remains unknown. In the present study, we found that miR-128b expression was down-regulated in tissues from 18 GC patients and 3 carcinoma cell lines. In turn, over-expression of miR-128b suppressed GC cell proliferation, invasion and promoted apoptosis. Moreover, miR-128b was predicted to bind the 3'UTR of PDK1 gene using bioinformatic target-screening tools. Accordingly, ectopic expression of miR-128b inhibited the PDK1 expression at both transcriptional and post-transcriptional levels, and furthermore, the expression of gene tailed by the 3′UTR of PDK1 gene was significantly decreased in a dualluciferase reporter assay, suggesting that PDK1 was a direct target of miR-128b in GC cells. In the conditon of miR-128b over-expression, we also observed spontaneous inactivation of the Akt/NF-κB signalling, implying PDK1 was a potential regulator of this pathway. In conclusion, our study shed some novel light on miR-128b-PDK1/Akt/NF-κB axis on GC progression.

  11. miR-208-3p promotes hepatocellular carcinoma cell proliferation and invasion through regulating ARID2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Peng; Wu, Dingguo; You, Yu; Sun, Jing; Lu, Lele; Tan, Jiaxing; Bie, Ping, E-mail: bieping2010@163.com

    2015-08-15

    MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level. miRNA dysregulation plays a causal role in cancer progression. In this study, miR-208-3p was highly expressed and directly repressed ARID2 expression. As a result, ARID2 expression in hepatocellular carcinoma (HCC) was decreased. In vitro, miR-208-3p down-regulation and ARID2 over-expression elicited similar inhibitory effects on HCC cell proliferation and invasion. In vivo test results revealed that miR-208-3p down-regulation inhibited HCC tumorigenesis in Hep3B cells. Moreover, ARID2 was possibly a downstream element of transforming growth factor beta1 (TGFβ1)/miR-208-3p/ARID2 regulatory pathway. These findings suggested that miR-208-3p up-regulation is associated with HCC cell progression and may provide a new target for liver cancer treatment. - Highlights: • miR-208-3p was highly expressed and directly repressed the expression of ARID2 in HCC. • miR-208-3p contributed to HCC cell progression both in vitro and in vivo. • Over-expression of ARID2 inhibited the HCC cell proliferation and invasion. • Restoration of ARID2 partly reversed the the effect of miR-208-3p down-regulation on HCC cells. • Newly regulatory pathway: miR-208-3p mediated the repression of ARID2 by TGFβ1 in HCC cells.

  12. Reduced expression of brain-enriched microRNAs in glioblastomas permits targeted regulation of a cell death gene.

    Directory of Open Access Journals (Sweden)

    Rebecca L Skalsky

    Full Text Available Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers.

  13. Regulation of copper homeostasis and biotic interactions by microRNA 398b in common bean.

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    Loreto Naya

    Full Text Available MicroRNAs are recognized as important post-transcriptional regulators in plants. Information about the roles of miRNAs in common bean (Phaseolus vulgaris L., an agronomically important legume, is yet scant. The objective of this work was to functionally characterize the conserved miRNA: miR398b and its target Cu/Zn Superoxide Dismutase 1 (CSD1 in common bean. We experimentally validated a novel miR398 target: the stress up-regulated Nodulin 19 (Nod19. Expression analysis of miR398b and target genes -CSD1 and Nod19- in bean roots, nodules and leaves, indicated their role in copper (Cu homeostasis. In bean plants under Cu toxicity miR398b was decreased and Nod19 and CSD1, that participates in reactive oxygen species (ROS detoxification, were up-regulated. The opposite regulation was observed in Cu deficient bean plants; lower levels of CSD1 would allow Cu delivery to essential Cu-containing proteins. Composite common bean plants with transgenic roots over-expressing miR398 showed ca. 20-fold higher mature miR398b and almost negligible target transcript levels as well as increased anthocyanin content and expression of Cu-stress responsive genes, when subjected to Cu deficiency. The down-regulation of miR398b with the consequent up-regulation of its targets was observed in common bean roots during the oxidative burst resulting from short-time exposure to high Cu. A similar response occurred at early stage of bean roots inoculated with Rhizobium tropici, where an increase in ROS was observed. In addition, the miR398b down-regulation and an increase in CSD1 and Nod19 were observed in bean leaves challenged with Sclerotinia scleortiorum fungal pathogen. Transient over-expression of miR398b in Nicotiana benthamiana leaves infected with S. sclerotiorum resulted in enhanced fungal lesions. We conclude that the miR398b-mediated up-regulation of CSD and Nod19 is relevant for common bean plants to cope with oxidative stress generated in abiotic and biotic

  14. MBD5 regulates iron metabolism via methylation-independent genomic targeting of Fth1 through KAT2A in mice.

    Science.gov (United States)

    Tao, Yunlong; Wu, Qian; Guo, Xin; Zhang, Zhuzhen; Shen, Yuanyuan; Wang, Fudi

    2014-07-01

    Ferritin plays important roles in iron metabolism and controls iron absorption in the intestine. The ferritin subunits ferritin heavy chain (Fth1) and ferritin light chain (Ftl1) are tightly regulated at both the transcriptional and post-transcriptional levels. However, mechanisms of maintaining stable, basal expression of Fth1 are poorly understood. Here, we show that global deletion of Mbd5 in mice induces an iron overload phenotype. Liver and serum iron levels in Mbd5(-/-) mice were 3·2-fold and 1·5-fold higher respectively, than wild-type littermates; moreover, serum ferritin was increased >5-fold in the Mbd5(-/-) mice. Mbd5 encodes a member of the methyl-CpG binding domain family; however, the precise function of this gene is poorly understood. Here, we found that intestinal Fth1 mRNA levels were decreased in Mbd5(-/-) mice. Loss of Fth1 expression in the intestine could lead to iron over-absorption. Furthermore, deleting Mbd5 specifically in the intestine resulted in a phenotype similar to that of conditional deletion of Fth1 mice. An Fth1 promoter-report luciferase assay indicated that overexpression of Mbd5 enhanced Fth1 transcription in a dose-dependent manner. Histone H4 acetylation of the Fth1 promoter was reduced in the intestine of Mbd5(-/-) mice and further analysis showed that histone acetyltransferase KAT2A was essential for MBD5-induced Fth1 transcription.

  15. HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes

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    Apostolia Guialis

    2013-10-01

    Full Text Available Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO mouse embryonic fibroblast (MEF cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle indicating their concerted function in mRNA metabolism.

  16. RuBisCO in Non-Photosynthetic Alga Euglena longa: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation.

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    Kristína Záhonová

    Full Text Available Euglena longa, a close relative of the photosynthetic model alga Euglena gracilis, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (rbcL. Here we provide new data illuminating the putative role of RuBisCO in E. longa. We demonstrated that the E. longa RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to E. gracilis, E. longa harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in E. longa, but their abundance is very low compared to E. gracilis. No RBCS monomers could be detected in E. longa, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown E. gracilis, but in E. longa, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct of RuBisCO in E. longa and suggest that its biological role in this species may be rather unorthodox, if any.

  17. RuBisCO in Non-Photosynthetic Alga Euglena longa: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation

    Science.gov (United States)

    Záhonová, Kristína; Füssy, Zoltán; Oborník, Miroslav; Eliáš, Marek

    2016-01-01

    Euglena longa, a close relative of the photosynthetic model alga Euglena gracilis, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (rbcL). Here we provide new data illuminating the putative role of RuBisCO in E. longa. We demonstrated that the E. longa RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to E. gracilis, E. longa harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS) as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in E. longa, but their abundance is very low compared to E. gracilis. No RBCS monomers could be detected in E. longa, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown E. gracilis, but in E. longa, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct) of RuBisCO in E. longa and suggest that its biological role in this species may be rather unorthodox, if any. PMID:27391690

  18. URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells.

    Science.gov (United States)

    Mita, Paolo; Savas, Jeffrey N; Briggs, Erica M; Ha, Susan; Gnanakkan, Veena; Yates, John R; Robins, Diane M; David, Gregory; Boeke, Jef D; Garabedian, Michael J; Logan, Susan K

    2016-12-02

    URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

  19. The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway.

    Science.gov (United States)

    Coyle, John H; Bor, Yeou-Cherng; Rekosh, David; Hammarskjold, Marie-Louise

    2011-07-01

    Post-transcriptional regulation of mRNA includes restriction mechanisms to prevent export and expression of mRNAs that are incompletely spliced. Here we present evidence that the mammalian protein Tpr is involved in this restriction. To study the role of Tpr in export of mRNA with retained introns, we used reporters in which the mRNA was exported either via the Nxf1/Nxt1 pathway using a CTE or via the Crm1 pathway using Rev/RRE. Our data show that even modest knockdown of Tpr using RNAi leads to a significant increase in export and translation from the mRNA containing the CTE. In contrast, Tpr perturbation has no effect on export of mRNA containing the RRE, either in the absence or presence of Rev. Also, no effects were observed on export of a completely spliced mRNA. Taken together, our results indicate that Tpr plays an important role in quality control of mRNA trafficked on the Nxf1 pathway.

  20. Regulation of deoxycytidine kinase expression and sensitivity to gemcitabine by micro-RNA 330 and promoter methylation in cancer cells.

    Science.gov (United States)

    Hodzic, Jasmina; Giovannetti, Elisa; Diosdado, Begoňa; Calvo, Begona Diosdado; Adema, A D; Peters, G J

    2011-12-01

    Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation. We hypothesized that dCK could be regulated by two additional mechanisms: micro-RNA (miRNA) and promoter methylation. Methylation-specific PCR (MSP) revealed methylation of the 3' GC box in three out of six cancer cell lines. The 3' GC box is located at the dCK promoter region. The methylation status was related to dCK mRNA expression. TargetScan and miRanda prediction algorithms revealed several possible miRNAs targeting dCK and identified miR-330 (micro-RNA 330) as the one conserved between the human, the chimpanzee, and the rhesus monkey genomes. Expression of miR-330 in various colon and lung cancer cell lines, as measured by QRT-PCR, varied five-fold between samples and correlated with in-vitro gemcitabine resistance (R = 0.82, p = 0.04). Exposure to gemcitabine also appeared to influence miR-330 levels in these cell lines. Furthermore, in our cell line panel, miR-330 expression negatively correlated with dCK mRNA expression (R = 0.74), suggesting a role of miR-330 in post-transcriptional regulation of dCK. In conclusion, the 3' GC box and miR-330 may regulate dCK expression in cancer cells.

  1. Altered regulation of ELAVL1/HuR in HLA-B27-expressing U937 monocytic cells.

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    Anna S Sahlberg

    Full Text Available OBJECTIVE: To investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP Embryonic Lethal Abnormal Vision (ELAV L1/Human antigen R (HuR expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα important in inflammatory response. METHODS: U937 monocytic cells were stably transfected with pSV2neo resistant vector (mock, wild type HLA-B27, or mutated HLA-B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2 were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA. RESULTS: Full length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC. Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27. CONCLUSION: Results show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

  2. Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system

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    Andrew Charles Penn

    2013-04-01

    Full Text Available The fine control of molecules mediating communication in the nervous system is key to adjusting neuronal responsiveness during development and in maintaining the stability of established networks in the face of altered sensory input. To prevent culmination of pathological recurrent network excitation or debilitating periods of quiescence, adaptive alterations occur in the signalling molecules and ion channels that control membrane excitability and synaptic transmission. However, rather than encoding (and thus ‘hardwiring’ modified gene copies, the nervous systems of metazoa have opted for expanding on post-transcriptional pre-mRNA splicing by altering key encoded amino acids using a conserved mechanism of A-to-I RNA editing: the enzymatic deamination of adenosine resulting in a change in the nucleotide to inosine. Inosine exhibits similar base-pairing properties to guanosine with respect to tRNA codon recognition, replication by polymerases and RNA secondary structure forming capacity. In addition to recoding within the open reading frame, adenosine deamination also occurs with high frequency throughout the non-coding transcriptome, where it affects multiple aspects of RNA metabolism and gene expression. We will describe here the recoding function of key RNA editing targets in the mammalian central nervous system (CNS and their potential to be regulated. We will then discuss how interactions of A-to-I editing with gene expression and alternative splicing could play a wider role in regulating the neuronal transcriptome. Finally, we will highlight the increasing complexity of this multifaceted control hub by summarising new findings from high-throughput studies.

  3. Betaine supplementation in maternal diet modulates the epigenetic regulation of hepatic gluconeogenic genes in neonatal piglets.

    Science.gov (United States)

    Cai, Demin; Jia, Yimin; Song, Haogang; Sui, Shiyan; Lu, Jingyu; Jiang, Zheng; Zhao, Ruqian

    2014-01-01

    In this study, gestational sows were fed control or betaine-supplemented diets (3 g/kg) throughout the pregnancy, and the newborn piglets were used to elucidate whether maternal dietary betaine affected offspring hepatic gluconeogenic genes through epigenetic mechanisms. Neonatal piglets born to betaine-supplemented sows had significantly higher serum and hepatic betaine contents, together with significantly greater expression of methionine metabolic enzymes in the liver. Interestingly, significantly higher serum concentrations of lactic acid and glucogenic amino acids, including serine, glutamate, methionine and histidine, were detected in the piglets born to betaine-supplemented sows, which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity, as well as greater protein expression of gluconeogenic enzymes, pyruvate carboxylase (PC), cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK1), mitochondrional phosphoenolpyruvate carboxykinase (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1). Moreover, maternal betaine significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes. The lower PEPCK1 mRNA was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3, while the up-regulated PEPCK2 and FBP1 mRNA was associated with DNA hypomethylation and more enriched activation histone mark H3K4me3. Furthermore, the expression of two miRNAs predicted to target PC and 6 miRNAs predicted to target PEPCK1 was dramatically suppressed in the liver of piglets born to betaine-supplemented sows. Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through enhanced hepatic methionine metabolism and epigenetic regulations, which involve DNA and histone methylations, and possibly miRNAs-mediated post-transcriptional mechanism.

  4. Adaptive Remodeling of the Bacterial Proteome by Specific Ribosomal Modification Regulates Pseudomonas Infection and Niche Colonisation.

    Science.gov (United States)

    Little, Richard H; Grenga, Lucia; Saalbach, Gerhard; Howat, Alexandra M; Pfeilmeier, Sebastian; Trampari, Eleftheria; Malone, Jacob G

    2016-02-01

    Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

  5. Wnt3a regulates tumor necrosis factor-α-stimulated interleukin-6 release in osteoblasts.

    Science.gov (United States)

    Natsume, Hideo; Tokuda, Haruhiko; Adachi, Seiji; Matsushima-Nishiwaki, Rie; Kato, Kenji; Minamitani, Chiho; Otsuka, Takanobu; Kozawa, Osamu

    2011-01-01

    It is recognized that Wnt pathways regulate bone metabolism. We have previously shown that tumor necrosis factor-α (TNF-α) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase)/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TNF-α-stimulated IL-6 synthesis in these cells. Wnt3a, which alone did not affect the IL-6 levels, significantly suppressed the TNF-α-stimulated IL-6 release. Lithium Chloride (LiCl), which is an inhibitor of GSK3β, markedly reduced the TNF-α-stimulated IL-6 release, similar to the results with Wnt3a. The suppression by Wnt3a or LiCl was also observed in the intracellular protein levels of IL-6 elicited by TNF-α. Wnt3a failed to affect the TNF-α-induced phosphorylation of p44/p42 MAP kinase, Akt, IκB or NFκB. Either Wnt3a or LiCl failed to reduce, rather increased the IL-6 mRNA expression stimulated by TNF-α. Lactacystin, a proteasome inhibitor, and bafilomycin A1, a lysosomal protease inhibitor, significantly restored the suppressive effect of Wnt3a on TNF-α-stimulated IL-6 release. Taken together, our results strongly suggest that Wnt3a regulates IL-6 release stimulated by TNF-α at post-transcriptional level in osteoblasts.

  6. Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

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    Ward Michael

    2009-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1 is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into

  7. Aquaporin 8 expression is reduced and regulated by microRNAs in patients with ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    MIN Min; PENG Li-hua; SUN Gang; GUO Ming-zhou; QIU Ze-wu; YANG Yun-sheng

    2013-01-01

    Background Ulcerative colitis (UC) is associated with differential expression of genes involved in inflammation and tissue remodeling.MicroRNA (miRNA) plays an important role in the pathogenesis of UC by regulating the gene expression at the post-transcriptional level and control crucial physiological processes.This study aimed to identify aquaporin 8 (AQP8) expression and its relationship with miRNA in UC patients.Methods Human colon samples,in this study,were obtained from 20 patients with UC and 16 healthy subjects undergoing diagnostic colonoscopy at the Chinese People's Liberation Army General Hospital between December 2009and June 2010.We screened different genes from UC tissues and healthy subjects using genome-wide microarray,quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting.Regulation of gene expression by miRNAs was assessed by luciferase reporter construct assays and transfection of specific miRNA mimics and inhibitor.Results We identified that 1596 genes were increased and 1301 genes were decreased in UC patients compared to healthy subjects.Among them,we focused on the analysis of AQP8 which was decreased three folds in UC tissues (P <0.01).The expression of AQP8 mRNA and protein were decreased in UC tissue and tumor necrosis factor (TNF)-αtreated HT29 cells compared with controls (P <0.05).We searched candidate target miRNAs of AQP8 through bioformatics and the luciferase report assay analysis indicated that miR-424,miR-195,miR-330,miR-612,and miR-16 which has complementary site in the 3-untranslated region (3'UTR) of AQP8 could decrease the relative luciferase activities by 10%-45%.Conclusion AQP8 and its relationship with miRNAs may be involved in the pathogenesis of UC.

  8. Emerging Perspectives on the Mechanisms,Regulation, and Distribution of Light Color Acclimation in Cyanobacteria

    Institute of Scientific and Technical Information of China (English)

    Andrian Gutu; David M. Kehoe

    2012-01-01

    Chromatic acclimation (CA) provides many cyanobacteria with the ability to tailor the properties of their lightharvesting antennae to the spectral distribution of ambient light.CA was originally discovered as a result of its dramatic cellular phenotype in red and green light.However,discoveries over the past decade have revealed that many pairs of light colors,ranging from blue to infrared,can trigger CA responses.The capacity to undergo CA is widespread geographically,occurs in most habitats around the world,and is found within all major cyanobacterial groups.In addition,many other cellular activities have been found to be under CA control,resulting in distinct physiological and morphological states for cells under different light-color conditions.Several types of CA appear to be the result of convergent evolution,where different strategies are used to achieve the final goal of optimizing light-harvesting antenna composition to maximize photon capture.The regulation of CA has been found to occur primarily at the level of RNA abundance.The CA-regulatory pathways uncovered thus far are two-component systems that use phytochrome-class photoreceptors with sensor-kinase domains to control response regulators that function as transcription factors.However,there is also at least one CAregulatory pathway that operates at the post-transcriptional level.It is becoming increasingly clear that large numbers of cyanobacterial species have the capacity to acclimate to a wide variety of light colors through the use of a range of different CA processes.

  9. Adaptive Remodeling of the Bacterial Proteome by Specific Ribosomal Modification Regulates Pseudomonas Infection and Niche Colonisation.

    Directory of Open Access Journals (Sweden)

    Richard H Little

    2016-02-01

    Full Text Available Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG. Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

  10. PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells.

    Science.gov (United States)

    Vaid, Samir; Ariz, Mohd; Chaturbedi, Amaresh; Kumar, Ganga Anil; Subramaniam, Kuppuswamy

    2013-04-01

    Signals that promote germ cell self-renewal by preventing premature meiotic entry are well understood. However, signals that control mitotic proliferation to promote meiotic differentiation have not been well characterized. In Caenorhabditis elegans, GLP-1 Notch signalling promotes the proliferative fate by preventing premature meiotic entry. The germline niche cell, which is the source of the ligand for GLP-1, spatially restricts GLP-1 signalling and thus enables the germ cells that have moved away from the niche to enter meiosis. Here, we show that the suppression of RAS/MAP kinase signalling in the mitotic and meiotic-entry regions is essential for the regulation of the mitosis-meiosis switch by niche signalling. We provide evidence that the conserved PUF family RNA-binding protein PUF-8 and the RAS GAP protein GAP-3 function redundantly to suppress the LET-60 RAS in the mitotic and meiotic entry regions. Germ cells missing both PUF-8 and GAP-3 proliferate in an uncontrolled fashion and fail to undergo meiotic development. MPK-1, the MAP kinase downstream of the LET-60 RAS, is prematurely activated in these cells; downregulation of MPK-1 activation eliminates tumours and restores differentiation. Our results further reveal that PUF-8 negatively regulates LET-60 expression at a post-transcriptional step. LET-60 is misexpressed in the puf-8(-) mutant germlines and PUF-8 physically interacts with the let-60 3' UTR. Furthermore, PUF-8 suppresses let-60 3' UTR-mediated expression in the germ cells that are transitioning from the mitotic to meiotic fate. These results reveal that PUF-8-mediated inhibition of the RAS/MAPK pathway is essential for mitotic-to-meiotic fate transition.

  11. Expression, regulation and clinical relevance of the ATPase inhibitory factor 1 in human cancers

    Science.gov (United States)

    Sánchez-Aragó, M; Formentini, L; Martínez-Reyes, I; García-Bermudez, J; Santacatterina, F; Sánchez-Cenizo, L; Willers, I M; Aldea, M; Nájera, L; Juarránz, Á; López, E C; Clofent, J; Navarro, C; Espinosa, E; Cuezva, J M

    2013-01-01

    Recent findings in colon cancer cells indicate that inhibition of the mitochondrial H+-adenosine triphosphate (ATP) synthase by the ATPase inhibitory factor 1 (IF1) promotes aerobic glycolysis and a reactive oxygen species (ROS)-mediated signal that enhances proliferation and cell survival. Herein, we have studied the expression, biological relevance, mechanism of regulation and potential clinical impact of IF1 in some prevalent human carcinomas. We show that IF1 is highly overexpressed in most (>90%) of the colon (n=64), lung (n=30), breast (n=129) and ovarian (n=10) carcinomas studied as assessed by different approaches in independent cohorts of cancer patients. The expression of IF1 in the corresponding normal tissues is negligible. By contrast, the endometrium, stomach and kidney show high expression of IF1 in the normal tissue revealing subtle differences by carcinogenesis. The overexpression of IF1 also promotes the activation of aerobic glycolysis and a concurrent ROS signal in mitochondria of the lung, breast and ovarian cancer cells mimicking the activity of oligomycin. IF1-mediated ROS signaling activates cell-type specific adaptive responses aimed at preventing death in these cell lines. Remarkably, regulation of IF1 expression in the colon, lung, breast and ovarian carcinomas is exerted at post-transcriptional levels. We demonstrate that IF1 is a short-lived protein (t1/2 ∼100 min) strongly implicating translation and/or protein stabilization as main drivers of metabolic reprogramming and cell survival in these human cancers. Analysis of tumor expression of IF1 in cohorts of breast and colon cancer patients revealed its relevance as a predictive marker for clinical outcome, emphasizing the high potential of IF1 as therapeutic target. PMID:23608753

  12. Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis

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    van Helden Paul

    2010-05-01

    observed, however, our results indicate that an additional regulatory mechanism may be involved. NADP+-GDH seemed to be involved in nitrogen assimilation due to a constitutive aminating activity. The deaminating reaction, however was observed to change in response to varying ammonium concentrations which suggests that NADP+-GDH is also regulated in response to nitrogen availability. The regulation of NADP+-GDH activity was not reflected at the level of gene transcription thereby implicating post-transcriptional modification as a regulatory mechanism in response to nitrogen availability.

  13. Plant phosphorus acquisition in a common mycorrhizal network: regulation of phosphate transporter genes of the Pht1 family in sorghum and flax.

    Science.gov (United States)

    Walder, Florian; Brulé, Daphnée; Koegel, Sally; Wiemken, Andres; Boller, Thomas; Courty, Pierre-Emmanuel

    2015-03-01

    In a preceding microcosm study, we found huge differences in phosphorus (P) acquisition in sorghum (Sorghum bicolor) and flax (Linum usitatissimum) sharing a common mycorrhizal network (CMN). Is the transcriptional regulation of arbuscular mycorrhizal (AM)-induced inorganic orthophosphate (Pi) transporters responsible for these differences? We characterized and analyzed the expression of Pi transporters of the Pht1 family in both plant species, and identified two new AM-inducible Pi transporters in flax. Mycorrhizal Pi acquisition was strongly affected by the combination of plant and AM fungal species. A corresponding change in the expression of two AM-inducible Pht1 transporters was noticed in both plants (SbPT9, SbPT10, LuPT5 and LuPT8), but the effect was very weak. Overall, the expression level of these genes did not explain why flax took up more Pi from the CMN than did sorghum. The post-transcriptional regulation of the transporters and their biochemical properties may be more important for their function than the fine-tuning of their gene expression.

  14. Pseudogene OCT4-pg4 functions as a natural micro RNA sponge to regulate OCT4 expression by competing for miR-145 in hepatocellular carcinoma.

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    Wang, Lei; Guo, Zhang-Yan; Zhang, Rui; Xin, Bo; Chen, Rui; Zhao, Jing; Wang, Tao; Wen, Wei-Hong; Jia, Lin-Tao; Yao, Li-Bo; Yang, An-Gang

    2013-08-01

    The POU transcription factor OCT4 is a pleiotropic regulator of gene expression in embryonic stem cells. Recent studies demonstrated that OCT4 is aberrantly expressed in multiple types of human cancer; however, the underlying molecular mechanism remains largely unknown. In this study, we report that OCT4-pg4, a pseudogene of OCT4, is abnormally activated in hepatocellular carcinoma (HCC). The expression level of OCT4-pg4 is positively correlated with that of OCT4, and both gene transcripts can be directly targeted by a tumor-suppressive micro RNA miR-145. We find that the non-coding RNA OCT4-pg4 is biologically active, as it can upregulate OCT4 protein level in HCC. Mechanistic analysis revealed that OCT4-pg4 functions as a natural micro RNA sponge to protect OCT4 transcript from being inhibited by miR-145. In addition, our study also showed that OCT4-pg4 can promote growth and tumorigenicity of HCC cells, thus exerting an oncogenic role in hepatocarcinogenesis. Furthermore, survival analysis suggests that high OCT4-pg4 level is significantly correlated with poor prognosis of HCC patients. Taken together, our finding adds a new layer of post-transcriptional regulation of OCT4 and sheds new light on the treatment of human HCC.

  15. MicroRNA与肿瘤糖代谢研究进展%Research progress in microRNA regulation of tumor glucose metabolism

    Institute of Scientific and Technical Information of China (English)

    胡骏; 殷保兵

    2013-01-01

    Aerobic glycolysis,which is also termed as Warburg effect,is one of the most common and profound biochemical phenotypes of tumor ceils.Several oncogenes and tumor suppressors genes have been implicated in aerobic glycolysis.MicroRNAs can control this metabolic switch by regulating oncogenes and tumor suppressors genes in a post-transcriptional way.A deeper understanding of the mechanisms and pathways by which miRNAs regulate the aerobic glycolysis will hopefully lead to a new therapeutic strategy for malignant cancer.%有氧糖酵解(Warburg效应)是肿瘤细胞的重要特征,它是癌基因和抑癌基因共同作用的结果.microRNA (miRNA)能够在转录后水平参与这些癌基因和抑癌基因的表达来调控肿瘤糖代谢方式的转变.对miRNA调控糖代谢机制的深入研究能够提供新的肿瘤治疗策略.

  16. The GATA factor elt-1 regulates C. elegans developmental timing by promoting expression of the let-7 family microRNAs.

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    Cohen, Max L; Kim, Sunhong; Morita, Kiyokazu; Kim, Seong Heon; Han, Min

    2015-03-01

    Postembryonic development in Caenorhabditis elegans is a powerful model for the study of the temporal regulation of development and for the roles of microRNAs in controlling gene expression. Stable switch-like changes in gene expression occur during development as stage-specific microRNAs are expressed and subsequently down-regulate other stage-specific factors, driving developmental progression. Key genes in this regulatory network are phylogenetically conserved and include the post-transcriptional microRNA repressor LIN-28; the nuclear hormone receptor DAF-12; and the microRNAs LIN-4, LET-7, and the three LET-7 family miRNAs (miR-48, miR-84, and miR-241). DAF-12 is known to regulate transcription of miR-48, miR-84 and miR-241, but its contribution is insufficient to account for all of the transcriptional regulation implied by the mutant phenotypes. In this work, the GATA-family transcription factor ELT-1 is identified from a genetic enhancer screen as a regulator of developmental timing in parallel to DAF-12, and is shown to do so by promoting the expression of the LET-7, miR-48, miR-84, and miR-241 microRNAs. The role of ELT-1 in developmental timing is shown to be separate from its role in cell-fate maintenance during post-embryonic development. In addition, analysis of Chromatin Immnoprecipitation (ChIP) data from the modENCODE project and this work suggest that the contribution of ELT-1 to the control of let-7 family microRNA expression is likely through direct transcription regulation.

  17. miR-29a modulates SCD expression and is regulated in response to a saturated fatty acids diet in juvenile GIFT (Oreochromis niloticus).

    Science.gov (United States)

    Qiang, Jun; Tao, Yi Fan; He, Jie; Sun, Yi Lan; Xu, Pao

    2017-02-06

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate target gene expression by binding to the 3'untranslated region (3'UTR) of the target mRNA. MiRNAs regulate a large variety of genes, including those involved in liver biology and disease. Here, we report for the first time that miR-29a post-transcriptionally regulates stearoyl-CoA desaturase (SCD) by binding to its 3'UTR in genetically improved farmed tilapia (GIFT), Oreochromis niloticus, as shown by a 3'UTR luciferase reporter assay. miR-29a antagomir treatment in vivo resulted in significant up-regulation of SCD expression. We found that miR-29a expression was negatively correlated with SCD expression in GIFT liver. Inhibition of miR-29a led to a significant increase in SCD expression on day 60 induced by a saturated fatty acids diet, thereby increasing conversion of 16:0 and 18:0 to 16:1 and 18:1 and activating serum insulin, which would favor glucose and lipid uptake by the liver. These results indicate that miR-29a regulates SCD levels by binding to its 3'UTR and this interaction affects saturated fatty acids stress induction and insulin and lipid accumulation in serum. Our results suggest that miR-29a is critical in regulating lipid metabolism homeostasis in GIFT liver and this might provide a basis for understanding the biological processes and therapeutic intervention encountered in fatty liver.

  18. The GATA factor elt-1 regulates C. elegans developmental timing by promoting expression of the let-7 family microRNAs.

    Directory of Open Access Journals (Sweden)

    Max L Cohen

    2015-03-01

    Full Text Available Postembryonic development in Caenorhabditis elegans is a powerful model for the study of the temporal regulation of development and for the roles of microRNAs in controlling gene expression. Stable switch-like changes in gene expression occur during development as stage-specific microRNAs are expressed and subsequently down-regulate other stage-specific factors, driving developmental progression. Key genes in this regulatory network are phylogenetically conserved and include the post-transcriptional microRNA repressor LIN-28; the nuclear hormone receptor DAF-12; and the microRNAs LIN-4, LET-7, and the three LET-7 family miRNAs (miR-48, miR-84, and miR-241. DAF-12 is known to regulate transcription of miR-48, miR-84 and miR-241, but its contribution is insufficient to account for all of the transcriptional regulation implied by the mutant phenotypes. In this work, the GATA-family transcription factor ELT-1 is identified from a genetic enhancer screen as a regulator of developmental timing in parallel to DAF-12, and is shown to do so by promoting the expression of the LET-7, miR-48, miR-84, and miR-241 microRNAs. The role of ELT-1 in developmental timing is shown to be separate from its role in cell-fate maintenance during post-embryonic development. In addition, analysis of Chromatin Immnoprecipitation (ChIP data from the modENCODE project and this work suggest that the contribution of ELT-1 to the control of let-7 family microRNA expression is likely through direct transcription regulation.

  19. Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland.

    Science.gov (United States)

    Maronde, Erik; Saade, Anastasia; Ackermann, Katrin; Goubran-Botros, Hany; Pagan, Cecile; Bux, Roman; Bourgeron, Thomas; Dehghani, Faramarz; Stehle, Jörg H

    2011-08-01

    Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

  20. CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs.

    Science.gov (United States)

    Bai, Ying; Lu, Huafei; Machida, Curtis A

    2006-10-01

    The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.

  1. Rapamycin (Sirolimus) alters mechanistic target of rapamycin pathway regulation and microRNA expression in mouse meiotic spermatocytes.

    Science.gov (United States)

    Mukherjee, A; Koli, S; Reddy, K V R

    2015-09-01

    target genes post-transcriptionally.

  2. Limb immobilization induces a coordinate down-regulation of mitochondrial and other metabolic pathways in men and women.

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    Arkan Abadi

    Full Text Available Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilization-induced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24 were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H and 14 days (14 D of immobilization. Muscle cross sectional area (approximately 5% and strength (10-20% were significantly reduced in men and women (approximately 5% and 10-20%, respectively after 14 D of immobilization. Micro-array analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly down-regulated and up-regulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and post-transcriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following short-term immobilization in humans.

  3. Differential regulation of LncRNA-SARCC suppresses VHL-mutant RCC cell proliferation yet promotes VHL-normal RCC cell proliferation via modulating androgen receptor/HIF-2α/C-MYC axis under hypoxia.

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    Zhai, W; Sun, Y; Jiang, M; Wang, M; Gasiewicz, T A; Zheng, J; Chang, C

    2016-09-15

    It is well established that hypoxia contributes to tumor progression in a hypoxia inducible factor-2α (HIF-2α)-dependent manner in renal cell carcinoma (RCC), yet the role of long noncoding RNAs (LncRNAs) involved in hypoxia-mediated RCC progression remains unclear. Here we demonstrate that LncRNA-SARCC (Suppressing Androgen Receptor in Renal Cell Carcinoma) is differentially regulated by hypoxia in a von Hippel-Lindau (VHL)-dependent manner both in RCC cell culture and clinical specimens. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells. Mechanism dissection reveals that LncRNA-SARCC can post-transcriptionally regulate androgen receptor (AR) by physically binding and destablizing AR protein to suppress AR/HIF-2α/C-MYC signals. In return, HIF-2α can transcriptionally regulate the LncRNA-SARCC expression via binding to hypoxia-responsive elements on the promoter of LncRNA-SARCC. The negative feedback modulation between LncRNA-SARCC/AR complex and HIF-2α signaling may then lead to differentially modulated RCC progression in a VHL-dependent manner. Together, these results may provide us a new therapeutic approach via targeting this newly identified signal from LncRNA-SARCC to AR-mediated HIF-2α/C-MYC signals against RCC progression.

  4. Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA 156 Module by the Homeodomain Proteins PENNYWISE and POUND-FOOLISH in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Shruti Lal; Leo Bryan Pacis; Harley M.S. Smith

    2011-01-01

    The morphology of inflorescences is regulated in part by the temporal and spatial events that regulate flower specification.In Arabidopsis,an endogenous flowering time pathway mediated by a subset of SQUAMOSA PROMOTERBINDING PROTEIN-LIKE (SPL) transcription factors,including SPL3,SPL4,and SPL5,function to specify flowers by activating floral meristem identity genes.During shoot development,SPL3,SPL4,and SPL5 are post-transcriptionally regulated by microRNA156 (miR156).The photoperiod regulated florigenic signal,FLOWERING LOCUS T (FT),promotes floral induction,in part by activating SPL3,SPL4,and SPL5.In turn,these SPLs function in parallel with FT to specify flower meristems.Two related BELL1-like homeobox genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) expressed in the shoot apical meristem are absolutely required for the specification of floral meristems.Genetic studies show that the floral specification function of FT depends upon PNY and PNF; however,the interplay between these homeodomain proteins and SPLs is not known.In this manuscript,we show that the photoperiodic floral induction of SPL3,SPL4,and SPL5 is dependent upon PNY and PNF.Further,PNY and PNF also control SPL3,SPL4,and SPL5 expression by negatively regulating miR156.Lastly,ectopic expression of SPL4 partially rescues the pny pnf non-flower-producing phenotype,while overexpression of SPL3 or SPL5 in pny pnf plants was unable to restore flower specification.These results suggest that:(1) SPL3,SPL4,and SPL5 function is dependent upon PNY and PNF,or (2) expression of multiple SPLs is required for floral specification in pny pnf plants.

  5. The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

    Science.gov (United States)

    Whisenant, Thomas C; Peralta, Eigen R; Aarreberg, Lauren D; Gao, Nina J; Head, Steven R; Ordoukhanian, Phillip; Williamson, Jamie R; Salomon, Daniel R

    2015-01-01

    Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA

  6. cGMP-independent nitric oxide signaling and regulation of the cell cycle

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    Cintron Ana

    2005-11-01

    Full Text Available Abstract Background Regulatory functions of nitric oxide (NO• that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO• on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1 were exposed to S-nitrosoglutathione, a NO• donor, or glutathione alone (control for 6 h without or with dibutyryl-cAMP (Bt2cAMP, and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO•-induced decreases in cAMP. Results NO• regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43% and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE. Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO•, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO• and Bt2cAMP effects showed that NO• regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO• annotated to G1/S (7/8 and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27; 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO•, as was E2F1 binding to E2F promoter elements. NO• activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M. Conclusion NO• coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO• may rely

  7. AB035. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator mir-27b in the spermatozoa of patients with asthenozoospermia

    Science.gov (United States)

    Zhou, Jun-Hao; Zhou, Qi-Zhao; Lyu, Xiao-Ming; Zhu, Ting; Chen, Zi-Jian; Chen, Ming-Kun; Xia, Hui; Wang, Chun-Yan; Qi, Tao; Li, Xin; Liu, Cun-Dong

    2016-01-01

    Background Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia Methods Spermatozoa were isolated from 90 study subjects’ ejaculated semen samples. DNA methylation was evaluated using bisulfite-sequencing PCR and methylation-specific PCR. The CRISP2 mRNA and protein expression levels were examined in the ejaculated spermatozoa by qRT-PCR and Western blot respectively. miRNA expression was detected by qRT-PCR. The direct regulatory effect of miR-27b on CRISP2 was predicted computationally and validated via luciferase reporter assay and in vitro experiments in which miR-27b mimic or inhibitor was transfected into 293T cells. Respective correlations of miR-27b and CRISP2 protein expression with clinical features were analyzed using Spearman’s correlation coefficient. Results Initially, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic males. Meanwhile, methylation was not found in CRISP2 promoter. These data suggest a possible post-transcriptional regulation of CRISP2 in asthenozoospermia. Subsequently, bioinformatics prediction, luciferase reporter assay and miR-27b transfection experiments revealed that miR-27b could specifically target CRISP2 by binding its 3’-UTR, suppressing CRISP2 expression post-transcriptionally. Further evidence was provided by the clinical observation of a high miR-27b expression in the ejaculated spermatozoa from asthenozoospermic patients and a negative correlation between miR-27b and CRISP2 protein expression. Finally, a retrospective

  8. miR-379 regulates cyclin B1 expression and is decreased in breast cancer.

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    Sonja Khan

    Full Text Available MicroRNAs are small non-coding RNA molecules that control gene expression post-transcriptionally, and are known to be altered in many diseases including breast cancer. The aim of this study was to determine the relevance of miR-379 in breast cancer. miR-379 expression was quantified in clinical samples including tissues from breast cancer patients (n=103, healthy controls (n=30 and patients with benign breast disease (n=35. The level of miR-379 and its putative target Cyclin B1 were investigated on all breast tissue specimens by RQ-PCR. Potential relationships with gene expression and patient clinicopathological details were also determined. The effect of miR-379 on Cyclin B1 protein expression and function was investigated using western blot, immunohistochemistry and proliferation assays respectively. Finally, the levels of circulating miR-379 were determined in whole blood from patients with breast cancer (n=40 and healthy controls (n=34. The level of miR-379 expression was significantly decreased in breast cancer (Mean(SEM 1.9 (0.09 Log10 Relative Quantity (RQ compared to normal breast tissues (2.6 (0.16 Log10 RQ, p<0.01. miR-379 was also found to decrease significantly with increasing tumour stage. A significant negative correlation was determined between miR-379 and Cyclin B1 (r=-0.31, p<0.001. Functional assays revealed reduced proliferation (p<0.05 and decreased Cyclin B1 protein levels following transfection of breast cancer cells with miR-379. Circulating miR-379 was not significantly dysregulated in patients with breast cancer compared to healthy controls (p=0.42. This data presents miR-379 as a novel regulator of Cyclin B1 expression, with significant loss of the miRNA observed in breast tumours.

  9. Complex regulation and multiple developmental functions of misfire, the Drosophila melanogaster ferlin gene

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    Wakimoto Barbara T

    2007-03-01

    Full Text Available Abstract Background Ferlins are membrane proteins with multiple C2 domains and proposed functions in Ca2+ mediated membrane-membrane interactions in animals. Caenorhabditis elegans has two ferlin genes, one of which is required for sperm function. Mammals have several ferlin genes and mutations in the human dysferlin (DYSF and otoferlin (OTOF genes result in muscular dystrophy and hearing loss, respectively. Drosophila melanogaster has a single ferlin gene called misfire (mfr. A previous study showed that a mfr mutation caused male sterility because of defects in fertilization. Here we analyze the expression and structure of the mfr gene and the consequences of multiple mutations to better understand the developmental function of ferlins. Results We show that mfr is expressed in the testis and ovaries of adult flies, has tissue-specific promoters, and expresses alternatively spliced transcripts that are predicted to encode distinct protein isoforms. Studies of 11 male sterile mutations indicate that a predicted Mfr testis isoform with five C2 domains and a transmembrane (TM domain is required for sperm plasma membrane breakdown (PMBD and completion of sperm activation during fertilization. We demonstrate that Mfr is not required for localization of Sneaky, another membrane protein necessary for PMBD. The mfr mutations vary in their effects in females, with a subset disrupting egg patterning and causing a maternal effect delay in early embryonic development. Locations of these mutations indicate that a short Mfr protein isoform carries out ferlin activities during oogenesis. Conclusion The mfr gene exhibits complex transcriptional and post-transcriptional regulation and functions in three developmental processes: sperm activation, egg patterning, and early embryogenesis. These functions are in part due to the production of protein isoforms that vary in the number of C2 domains. These findings help establish D. melanogaster as model system for

  10. IL-1β up-regulates expression of IL-8 in endometrial stromal cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhang Guiyu; Ren Shuwen; Zhang Youzhong; Yang Xingsheng

    2005-01-01

    Objective:To investigate the effects of interleukin-1beta (IL-1β) on expression of IL-8 in endometrial stromal cells (ESC) and evaluate the relationship between IL1 β and IL-8 ,and the significance of IL-1β in the development of endometriosis. Methods:The endometrial stromal cells obtained from patient with and without endometriosis cultured within 3 ~5 passage were exposed to various concentrations of IL-1β. The amount of IL-8 protein was assessed by ELISA. The expression of IL-8 mRNA was determined by RT-PCR. Results: 1. IL-8 protein was detected in culture supernatant of which the cells were not treated with IL-1β. The amount of IL-8 protein secretion increased obviously after stimulation with IL-1β at 1.0ng/ml for 4h and the peak of secretion was at 12h. 2. Expression of IL-8 mRNA was positive in unstimulated endometrial stromal cells. However, after stromal cells were incubated with IL-1β, the intensity of expression of IL-8 mRNA was obviously increased and demonstrated a dose-and timedependent manner. Increase of IL-8 mRNA was observed following stimulation with IL-1β for 4h ,and the peak at 12h. Conclusions:IL-1β induces endometrial stromal cell of endometriosis to express IL-8 not only at transcription level but also at post-transcription level. This up-regulation is dose-and time-dependent. IL-1β may play an important role in the onset of endometriosis.

  11. Differential regulation of genes encoding manganese peroxidase (MnP) in the basidiomycete Ceriporiopsis subvermispora.

    Science.gov (United States)

    Manubens, Augusto; Avila, Marcela; Canessa, Paulo; Vicuña, Rafael

    2003-09-01

    We previously identified and characterized three mnp genes coding for manganese peroxidase (MnP) in the white rot fungus Ceriporiopsis subvermispora. In this work, we assessed transcript levels of mnp genes in liquid cultures of this fungus grown under various conditions. In the absence of Mn(2+), mnp1 and mnp2 mRNA were detected by Northern hybridization, irrespective of the lack of extracellular MnP activity. Addition of Mn(2+) to the cultures led to a marked increase in both transcripts, the highest titers being observed at 10 micro M Mn(2+). mnp1 mRNA was not detected at Mn(2+ )concentrations above 80 micro M, whereas mnp2 mRNA was still observed at 320 micro M Mn(2+). Differential regulation of these genes was confirmed by the addition of Cu(2+), Zn(2+), Ag(+) and Cd(2+). These metal ions dramatically elevated both transcripts and also allowed the detection of the mnp3 transcript. In most cases, the increase in mRNA levels was partially abolished by the simultaneous presence of Mn(2+), although the latter was strictly required to detect extracellular MnP activity. However, the lignin-related compound syringic acid specifically increased the mnp1 transcript, although only in the absence of Mn(2+). These results indicate that there is no clear correlation between mnp mRNA levels and MnP activity. In addition, they strongly suggest that Mn(2+) plays a post-transcriptional role which is essential for the presence of active MnP in the extracellular fluid.

  12. The Fuzzy Logic of MicroRNA Regulation: A Key to Control Cell Complexity.

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    Ripoli, Andrea; Rainaldi, Giuseppe; Rizzo, Milena; Mercatanti, Alberto; Pitto, Letizia

    2010-08-01

    Genomic and clinical evidence suggest a major role of microRNAs (miRNAs) in the regulatory mechanisms of gene expression, with a clear impact on development and physiology; miRNAs are a class of endogenous 22-25 nt single-stranded RNA molecules, that negatively regulate gene expression post-transcriptionally, by imperfect base pairing with the 3' UTR of the corresponding mRNA target. Because of this imperfection, each miRNA can bind multiple targets, and multiple miRNAs can bind the same mRNA target; although digital, the miRNAs control mechanism is characterized by an imprecise action, naturally understandable in the theoretical framework of fuzzy logic.A major practical application of fuzzy logic is represented by the design and the realization of efficient and robust control systems, even when the processes to be controlled show chaotic, deterministic as well unpredictable, behaviours. The vagueness of miRNA action, when considered together with the controlled and chaotic gene expression, is a hint of a cellular fuzzy control system. As a demonstration of the possibility and the effectiveness of miRNA based fuzzy mechanism, a fuzzy cognitive map -a mathematical formalism combining neural network and fuzzy logic- has been developed to study the apoptosis/proliferation control performed by the miRNA-17-92 cluster/E2F1/cMYC circuitry.When experimentally demonstrated, the concept of fuzzy control could modify the way we analyse and model gene expression, with a possible impact on the way we imagine and design therapeutic intervention based on miRNA silencing.

  13. Dietary fish oil blocks carcinogen-induced down-regulation of colonic protein kinase C isozymes.

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    Jiang, Y H; Lupton, J R; Chapkin, R S

    1997-02-01

    In order to elucidate the influence of dietary constituents on colonic intracellular signal transduction, the effect of different fats on rat colonic epithelial protein kinase C (PKC) alpha (classical), delta (novel) and lambda-zeta (atypical) expression was determined in carcinogen-treated animals. Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil); plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (15 and 37 weeks) in a 2x2x2 factorial design. At 5 and 6 weeks of age, animals were injected s.c. with either AOM at a dose of 15 mg/kg body weight or saline once a week for 2 weeks and continued on the same diet until termination of the study. At 15 and 37 weeks after the second injection, 10 rats from each treatment group were killed. Colonic PKC alpha, delta and lambda-zeta steady-state protein and mRNA levels were determined using immunoblotting and relative quantitative polymerase chain reaction, respectively. Colonic mucosa from rats injected with AOM had significantly suppressed membrane and cytosolic PKC alpha and cytosolic lambda-zeta protein levels (P fish oil diets had significantly higher (P protein levels relative to animals fed corn oil diets. However, the effect of diet and AOM on the steady-state expression of PKC alpha, delta and zeta mRNA was not consistent with changes in the respective isozyme protein levels, suggesting regulation at the post-transcriptional level. These data demonstrate that dietary fish oil blocks the carcinogen-induced decrease in the steady-state levels of colonic mucosal PKC delta and lambda-zeta, which may in part explain why this fat source protects against colon cancer development.

  14. Both 5' and 3' flanks regulate Zebrafish brain-derived neurotrophic factor gene expression

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    Heinrich Gerhard

    2004-05-01

    Full Text Available Abstract Background Precise control of developmental and cell-specific expression of the brain-derived neurotrophic factor (BDNF gene is essential for normal neuronal development and the diverse functions of BDNF in the adult organism. We previously showed that the zebrafish BDNF gene has multiple promoters. The complexity of the promoter structure and the mechanisms that mediate developmental and cell-specific expression are still incompletely understood. Results Comparison of pufferfish and zebrafish BDNF gene sequences as well as 5' RACE revealed three additional 5' exons and associated promoters. RT-PCR with exon-specific primers showed differential developmental and organ-specific expression. Two exons were detected in the embryo before transcription starts. Of the adult organs examined, the heart expressed a single 5' exon whereas the brain, liver and eyes expressed four of the seven 5' exons. Three of the seven 5' exons were not detectable by RT-PCR. Injection of promoter/GFP constructs into embryos revealed distinct expression patterns. The 3' flank profoundly affected expression in a position-dependent manner and a highly conserved sequence (HCS1 present in 5' exon 1c in a dehancer-like manner. Conclusions The zebrafish BDNF gene is as complex in its promoter structure and patterns of differential promoter expression as is its murine counterpart. The expression of two of the promoters appears to be regulated in a temporally and/or spatially highly circumscribed fashion. The 3' flank has a position-dependent effect on expression, either by affecting transcription termination or post-transcriptional steps. HCS1, a highly conserved sequence in 5' exon 1c, restricts expression to primary sensory neurons. The tools are now available for detailed genetic and molecular analyses of zebrafish BDNF gene expression.

  15. Inhibition of expression in Escherichia coli of a virulence regulator MglB of Francisella tularensis using external guide sequence technology.

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    Gaoping Xiao

    Full Text Available External guide sequences (EGSs have successfully been used to inhibit expression of target genes at the post-transcriptional level in both prokaryotes and eukaryotes. We previously reported that EGS accessible and cleavable sites in the target RNAs can rapidly be identified by screening random EGS (rEGS libraries. Here the method of screening rEGS libraries and a partial RNase T1 digestion assay were used to identify sites accessible to EGSs in the mRNA of a global virulence regulator MglB from Francisella tularensis, a Gram-negative pathogenic bacterium. Specific EGSs were subsequently designed and their activities in terms of the cleavage of mglB mRNA by RNase P were tested in vitro and in vivo. EGS73, EGS148, and EGS155 in both stem and M1 EGS constructs induced mglB mRNA cleavage in vitro. Expression of stem EGS73 and EGS155 in Escherichia coli resulted in significant reduction of the mglB mRNA level coded for the F. tularensis mglB gene inserted in those cells.

  16. miR-211 is a pro-survival micro-RNA that regulates chop expression in a PERK-dependent manner

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    Chitnis, Nilesh; Pytel, Dariusz; Bobrovnikova-Marjon, Ekaterina; Pant, Dhruv; Zheng, Hui; Maas, Nancy; Frederick, Brian; Kushner, Jake A.; Chodosh, Lewis A.; Koumenis, Constantinos; Fuchs, Serge Y.; Diehl, J. Alan

    2012-01-01

    Micro-RNAs typically function at the level of post-transcriptional gene silencing within the cytoplasm; however increasing evidence suggests that they may also function in nuclear, Argonaut containing complexes, to directly repress target gene transcription. We have investigated the role of micro-RNAs in mediating endoplasmic reticulum (ER) stress responses. ER stress triggers the activation of three signaling molecules: Ire-1α/β, PERK and ATF6 whose function is to facilitate adaption to the ensuing stress. We demonstrate that PERK induces miR-211, which in turn attenuates stress-dependent expression of the pro-apoptotic transcription factor chop/gadd153. MiR-211 directly targets the proximal chop/gadd153 promoter where it increases histone methylation and represses chop expression. Maximal chop accumulation ultimately correlates with miR-211 down regulation. Our data suggests a model where PERK-dependent miR-211 induction prevents premature chop accumulation and thereby provides a window of opportunity for the cell to re-establish homeostasis prior to apoptotic commitment. PMID:23022383

  17. The carbon storage regulator (Csr system exerts a nutrient-specific control over central metabolism in Escherichia coli strain Nissle 1917.

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    Olga Revelles

    Full Text Available The role of the post-transcriptional carbon storage regulator (Csr system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the csrA51 mutant affects the growth efficiency on a broad range of physiologically relevant carbon sources, including compounds utilized by the Entner-Doudoroff (ED pathway. Detailed investigations of the metabolomes and fluxomes of mutants and wild-type cells grown on carbon sources representative of glycolysis and of the ED pathway (glucose and gluconate, respectively, revealed significant re-adjusting of central carbon metabolism for both compounds in the csrA51 mutant. However, the metabolic re-adjusting observed on gluconate was strikingly different from that observed on glucose, indicating a nutrient-specific control of metabolism by the Csr system.

  18. The carbon storage regulator (Csr) system exerts a nutrient-specific control over central metabolism in Escherichia coli strain Nissle 1917.

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    Revelles, Olga; Millard, Pierre; Nougayrède, Jean-Philippe; Dobrindt, Ulrich; Oswald, Eric; Létisse, Fabien; Portais, Jean-Charles

    2013-01-01

    The role of the post-transcriptional carbon storage regulator (Csr) system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations) showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the csrA51 mutant affects the growth efficiency on a broad range of physiologically relevant carbon sources, including compounds utilized by the Entner-Doudoroff (ED) pathway. Detailed investigations of the metabolomes and fluxomes of mutants and wild-type cells grown on carbon sources representative of glycolysis and of the ED pathway (glucose and gluconate, respectively), revealed significant re-adjusting of central carbon metabolism for both compounds in the csrA51 mutant. However, the metabolic re-adjusting observed on gluconate was strikingly different from that observed on glucose, indicating a nutrient-specific control of metabolism by the Csr system.

  19. miR-122 regulates p53/Akt signalling and the chemotherapy-induced apoptosis in cutaneous T-cell lymphoma.

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    Valentina Manfè

    Full Text Available Advanced cutaneous T-cell lymphoma (CTCL is resistant to chemotherapy and presents a major area of medical need. In view of the known role of microRNAs (miRNAs in the regulation of cellular signalling, we aimed to identify the functionally important miRNA species, which regulate apoptosis in CTCL. Using a recently established model in which apoptosis of CTCL cell lines is induced by Notch-1 inhibition by γ-secretase inhibitors (GSIs, we found that miR-122 was significantly increased in the apoptotic cells. miR-122 up-regulation was not specific for GSI-1 but was also seen during apoptosis induced by chemotherapies including doxorubicin and proteasome blockers (bortezomib, MG132. miR-122 was not expressed in quiescent T-cells, but was detectable in CTCL: in lesional skin in mycosis fungoides and in Sézary cells purified from peripheral blood. In situ hybridization results showed that miR-122 was expressed in the malignant T-cell infiltrate and increased in the advanced stage mycosis fungoides. Surprisingly, miR-122 overexpression decreased the sensitivity to the chemotherapy-induced apoptosis via a signaling circuit involving the activation of Akt and inhibition of p53. We have also shown that induction of miR-122 occurred via p53 and that p53 post-transcriptionally up-regulated miR-122. miR-122 is thus an amplifier of the antiapoptotic Akt/p53 circuit and it is conceivable that a pharmacological intervention in this pathway may provide basis for novel therapies for CTCL.

  20. Differential regulation of pancreatic digestive enzymes during chronic high-fat diet-induced obesity in C57BL/6J mice.

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    Birk, Ruth Z; Rubio-Aliaga, Isabel; Boekschoten, Mark V; Danino, Hila; Müller, Michael; Daniel, Hannelore

    2014-07-28

    Exocrine pancreatic digestive enzymes are essential for the digestion of dietary components and are regulated by them. Chronic excess dietary high fat (HF) consumption is a contributing factor of diet-induced obesity (DIO) and associated chronic diseases and requires adaptation by the pancreas. The aim of the present study was to investigate the effects of chronic HF diet feeding on exocrine pancreatic digestive enzyme transcript levels in DIO C57BL/6J mice. C57BL/6J mice were fed diets containing either 10 or 45% energy (E%) derived from fat for 12 weeks (n 10 mice per diet group). Pancreatic tissue and blood samples were collected at 0, 4 and 12 weeks. The expression of a panel of exocrine pancreatic digestive enzymes was analysed using quantitative RT-PCR and Western blot analysis. The HF (45 E%) diet-fed C57BL/6J mice developed obesity, hyperleptinaemia, hyperglycaemia and hyperinsulinaemia. The transcript levels of pancreatic lipase (PL), pancreatic lipase-related protein 2 (PLRP2) and pancreatic phospholipase A2 (PLA2) were initially elevated; however, they were down-regulated to basal control levels at week 12. The transcript levels of colipase were significantly affected by diet and time. The protein levels of PL and PLRP2 responded to HF diet feeding. The transcript levels of amylase and proteases were not significantly affected by diet and time. The transcript levels of specific lipases in hyperinsulinaemic, hyperleptinaemic and hyperglycaemic DIO C57BL/6J mice are down-regulated. However, these mice compensate for this by the post-transcriptional regulation of the levels of proteins that respond to dietary fat. This suggests a complex regulatory mechanism involved in the modulation of fat digestion.

  1. miR-122 Regulates p53/Akt Signalling and the Chemotherapy-Induced Apoptosis in Cutaneous T-Cell Lymphoma

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    Manfè, Valentina; Biskup, Edyta; Rosbjerg, Anne; Kamstrup, Maria; Skov, Anne Guldhammer; Lerche, Catharina Margrethe; Lauenborg, Britt Thyssing; Ødum, Niels; Gniadecki, Robert

    2012-01-01

    Advanced cutaneous T-cell lymphoma (CTCL) is resistant to chemotherapy and presents a major area of medical need. In view of the known role of microRNAs (miRNAs) in the regulation of cellular signalling, we aimed to identify the functionally important miRNA species, which regulate apoptosis in CTCL. Using a recently established model in which apoptosis of CTCL cell lines is induced by Notch-1 inhibition by γ-secretase inhibitors (GSIs), we found that miR-122 was significantly increased in the apoptotic cells. miR-122 up-regulation was not specific for GSI-1 but was also seen during apoptosis induced by chemotherapies including doxorubicin and proteasome blockers (bortezomib, MG132). miR-122 was not expressed in quiescent T-cells, but was detectable in CTCL: in lesional skin in mycosis fungoides and in Sézary cells purified from peripheral blood. In situ hybridization results showed that miR-122 was expressed in the malignant T-cell infiltrate and increased in the advanced stage mycosis fungoides. Surprisingly, miR-122 overexpression decreased the sensitivity to the chemotherapy-induced apoptosis via a signaling circuit involving the activation of Akt and inhibition of p53. We have also shown that induction of miR-122 occurred via p53 and that p53 post-transcriptionally up-regulated miR-122. miR-122 is thus an amplifier of the antiapoptotic Akt/p53 circuit and it is conceivable that a pharmacological intervention in this pathway may provide basis for novel therapies for CTCL. PMID:22235305

  2. Insights from the Cold Transcriptome and Metabolome of Dendrobium officinale: Global Reprogramming of Metabolic and Gene Regulation Networks during Cold Acclimation

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    Wu, Zhi-Gang; Jiang, Wu; Chen, Song-Lin; Mantri, Nitin; Tao, Zheng-Ming; Jiang, Cheng-Xi

    2016-01-01

    Plant cold acclimation (CA) is a genetically complex phenomenon involving gene regulation and expression. Little is known about the cascading pattern of gene regulatroy network and the link between genes and metabolites during CA. Dendrobium officinale (DOKM) is an important medicinal and ornamental plant and hypersensitive to low temperature. Here, we used the large scale metabolomic and transcriptomic technologies to reveal the response to CA in DOKM seedlings based on the physiological profile analyses. Lowering temperature from 4 to –2°C resulted in significant increase (P antioxidant activities and electrolyte leakage (EL) during 24 h. The fitness CA piont of 0°C and control (20°C) during 20 h were firstly obtained according to physiological analyses. Subsequently, massive transcriptome and metabolome reprogramming occurred during CA. The gene to metabolite network demonstrated that the CA associated processes are highly energy demanding through activating hydrolysis of sugars, amino acids catabolism and citrate cycle. The expression levels of 2,767 genes were significantly affected by CA, including 153-fold upregulation of CBF transcription factor, 56-fold upregulation of MAPKKK16 protein kinase. Moreover, the gene interaction and regulation network analysis revealed that the CA as an active process, was regulated at the transcriptional, post-transcriptional, translational and post-translational levels. Our findings highligted a comprehensive regulatory mechanism including cold signal transduction, transcriptional regulation, and gene expression, which contributes a deeper understanding of the highly complex regulatory program during CA in DOKM. Some marker genes identified in DOKM seedlings will allow us to understand the role of each individual during CA by further functional analyses. PMID:27877182

  3. Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation

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    Fields Joshua A

    2012-10-01

    Full Text Available Abstract Background Although Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previously, we reported data illustrating the requirement for CsrA in several virulence related phenotypes of C. jejuni strain 81–176, indicating that the Csr pathway is important for Campylobacter pathogenesis. Results We compared the Escherichia coli and C. jejuni orthologs of CsrA and characterized the ability of the C. jejuni CsrA protein to functionally complement an E. coli csrA mutant. Phylogenetic comparison of E. coli CsrA to orthologs from several pathogenic bacteria demonstrated variability in C. jejuni CsrA relative to the known RNA binding domains of E. coli CsrA and in several amino acids reported to be involved in E. coli CsrA-mediated gene regulation. When expressed in an E. coli csrA mutant, C. jejuni CsrA succeeded in recovering defects in motility, biofilm formation, and cellular morphology; however, it failed to return excess glycogen accumulation to wild type levels. Conclusions These findings suggest that C. jejuni CsrA is capable of efficiently binding some E. coli CsrA binding sites, but not others, and provide insight into the biochemistry of C. jejuni CsrA.

  4. Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

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    Marc Chatenay-Lapointe

    Full Text Available Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

  5. Exercise, skeletal muscle and inflammation: ARE-binding proteins as key regulators in inflammatory and adaptive networks.

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    Beiter, Thomas; Hoene, Miriam; Prenzler, Frauke; Mooren, Frank C; Steinacker, Jürgen M; Weigert, Cora; Nieß, Andreas M; Munz, Barbara

    2015-01-01

    The role of inflammation in skeletal muscle adaptation to exercise is complex and has hardly been elucidated so far. While the acute inflammatory response to exercise seems to promote skeletal muscle training adaptation and regeneration, persistent, low-grade inflammation, as seen in a multitude of chronic diseases, is obviously detrimental. The regulation of cytokine production in skeletal muscle cells has been relatively well studied, yet little is known about the compensatory and anti-inflammatory mechanisms that resolve inflammation and restore tissue homeostasis. One important strategy to ensure sequential, timely and controlled resolution of inflammation relies on the regulated stability of mRNAs encoding pro-inflammatory mediators. Many key transcripts in early immune responses are characterized by the presence of AU-rich elements (AREs) in the 3'-untranslated regions of their mRNAs, allowing efficient fine-tuning of gene expression patterns at the post-transcriptional level. AREs exert their function by recruiting particular RNA-binding proteins, resulting, in most cases, in de-stabilization of the target transcripts. The best-characterized ARE-binding proteins are HuR, CUGBP1, KSRP, AUF1, and the three ZFP36 proteins, especially TTP/ZFP36. Here, we give a general introduction into the role of inflammation in the adaptation of skeletal muscle to exercise. Subsequently, we focus on potential roles of ARE-binding proteins in skeletal muscle tissue in general and specifically exercise-induced skeletal muscle remodeling. Finally, we present novel data suggesting a specific function of TTP/ZFP36 in exercise-induced skeletal muscle plasticity.

  6. MicroRNA-16 Modulates HuR Regulation of Cyclin E1 in Breast Cancer Cells

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    Xun Guo

    2015-03-01

    Full Text Available RNA binding protein (RBPs and microRNAs (miRNAs or miRs are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC. Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

  7. Effect of carbon source perturbations on transcriptional regulation of metabolic fluxes in Saccharomyces cerevisiae

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    Ülgen Kutlu Ö

    2007-03-01

    change in fluxes are correlating directly with the change in transcript levels of genes encoding their corresponding enzymes. For amino acid biosynthesis there was, however, not found to exist a similar correlation, and this may point to either post-transcriptional and/or metabolic regulation, or be due to the absence of a direct perturbation on the amino acid pathways in these experiments.

  8. Regulatory and functional interactions of plant growth regulators and plant glutathione S-transferases (GSTs).

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    Moons, Ann

    2005-01-01

    Plant glutathioneS-transferases (GSTs) are a heterogeneous superfamily of multifunctional proteins, grouped into six classes. The tau (GSTU) and phi (GSTF) class GSTs are the most represented ones and are plant-specific, whereas the smaller theta (GSTT) and zeta (GSTZ) classes are also found in animals. The lambda GSTs (GSTL) and the dehydroascorbate reductases (DHARs) are more distantly related. Plant GSTs perform a variety of pivotal catalytic and non-enzymatic functions in normal plant development and plant stress responses, roles that are only emerging. Catalytic functions include glutathione (GSH)-conjugation in the metabolic detoxification of herbicides and natural products. GSTs can also catalyze GSH-dependent peroxidase reactions that scavenge toxic organic hydroperoxides and protect from oxidative damage. GSTs can furthermore catalyze GSH-dependent isomerizations in endogenous metabolism, exhibit GSH-dependent thioltransferase safeguarding protein function from oxidative damage and DHAR activity functioning in redox homeostasis. Plant GSTs can also function as ligandins or binding proteins for phytohormones (i.e., auxins and cytokinins) or anthocyanins, thereby facilitating their distribution and transport. Finally, GSTs are also indirectly involved in the regulation of apoptosis and possibly also in stress signaling. Plant GST genes exhibit a diversity of expression patterns during biotic and abiotic stresses. Stress-induced plant growth regulators (i.e., jasmonic acid [JA], salicylic acid [SA], ethylene [ETH], and nitric oxide [NO] differentially activate GST gene expression. It is becoming increasingly evident that unique combinations of multiple, often interactive signaling pathways from various phytohormones and reactive oxygen species or antioxidants render the distinct transcriptional activation patterns of individual GSTs during stress. Underestimated post-transcriptional regulations of individual GSTs are becoming increasingly evident and roles

  9. Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3’UTR

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    Adel eDerghal

    2015-05-01

    Full Text Available The central nervous system monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3'-untranslated regions (3'UTR of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3’UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3'UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p and mir-488. We observed that mir-383, mir-384-3p and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus.

  10. Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3′UTR

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    Derghal, Adel; Djelloul, Mehdi; Airault, Coraline; Pierre, Clément; Dallaporta, Michel; Troadec, Jean-Denis; Tillement, Vanessa; Tardivel, Catherine; Bariohay, Bruno; Trouslard, Jérôme; Mounien, Lourdes

    2015-01-01

    The central nervous system (CNS) monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC) neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs) are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3′-untranslated regions (3′UTR) of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3′UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3′UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p, and mir-488. We observed that mir-383, mir-384-3p, and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p, and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p, and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus. PMID:25999818

  11. WNT7A Regulation by miR-15b in Ovarian Cancer.

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    James A MacLean

    Full Text Available WNT signaling is well known to play an important role in the regulation of development, cell proliferation and cell differentiation in a wide variety of normal and cancerous tissues. Despite the wealth of knowledge concerning when and where various WNT genes are expressed and downstream events under their control, there is surprisingly little published evidence of how they are regulated. We have recently reported that aberrant WNT7A is observed in serous ovarian carcinomas, and WNT7A is the sole ligand accelerating ovarian tumor progression through CTNNB1 (β-catenin/TCF signaling in the absence of CTNNB1 mutations. In the present study, we report that WNT7A is a direct target of miR-15b in ovarian cancer. We showed that a luciferase reporter containing the putative binding site of miR-15b in the WNT7A 3'-UTR was significantly repressed by miR-15b. Mutation of the putative binding site of miR-15b in the WNT7A 3'-UTR restored luciferase activity. Furthermore, miR-15b was able to repress increased levels of TOPFLASH activity by WNT7A, but not those induced by S33Y. Additionally, miR-15b dose-dependently decreased WNT7A expression. When we evaluated the prognostic impact of WNT7A and miR-15b expression using TCGA datasets, a significant inverse correlation in which high-expression of WNT7A and low-expression of miR-15b was associated with reduced survival rates of ovarian cancer patients. Treatment with decitabine dose-dependently increased miR-15b expression, and silencing of DNMT1 significantly increased miR-15b expression. These results suggest that WNT7A is post-transcriptionally regulated by miR-15b, which could be down-regulated by promoter hypermethylation, potentially via DNMT1, in ovarian cancer.

  12. Inter- and intra-combinatorial regulation by transcription factors and microRNAs

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    Chang Joseph T

    2007-10-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a novel class of non-coding small RNAs. In mammalian cells, miRNAs repress the translation of messenger RNAs (mRNAs or degrade mRNAs. miRNAs play important roles in development and differentiation, and they are also implicated in aging, and oncogenesis. Predictions of targets of miRNAs suggest that they may regulate more than one-third of all genes. The overall functions of mammalian miRNAs remain unclear. Combinatorial regulation by transcription factors alone or miRNAs alone offers a wide range of regulatory programs. However, joining transcriptional and post-transcriptional regulatory mechanisms enables higher complexity regulatory programs that in turn could give cells evolutionary advantages. Investigating coordinated regulation of genes by miRNAs and transcription factors (TFs from a statistical standpoint is a first step that may elucidate some of their roles in various biological processes. Results Here, we studied the nature and scope of coordination among regulators from the transcriptional and miRNA regulatory layers in the human genome. Our findings are based on genome wide statistical assessment of regulatory associations ("interactions" among the sets of predicted targets of miRNAs and sets of putative targets of transcription factors. We found that combinatorial regulation by transcription factor pairs and miRNA pairs is much more abundant than the combinatorial regulation by TF-miRNA pairs. In addition, many of the strongly interacting TF-miRNA pairs involve a subset of master TF regulators that co-regulate genes in coordination with almost any miRNA. Application of standard measures for evaluating the degree of interaction between pairs of regulators show that strongly interacting TF-miRNA, TF-TF or miRNA-miRNA pairs tend to include TFs or miRNAs that regulate very large numbers of genes. To correct for this potential bias we introduced an additional Bayesian measure that incorporates

  13. Regulation of miRNA in diabetes mellitus and its cardiovascular complications%miRNA对糖尿病及其心血管并发症的调控

    Institute of Scientific and Technical Information of China (English)

    徐凤; 钱玲玲; 王如兴

    2014-01-01

    miRNAs (microRNAs) are small RNA which regulate gene expressions at the post-transcriptional level.Recently studies have suggested that miRNA is associated with the development of diabetes mellitus and its complications.This artcle reviews the regulation of miRNA in diabetes mellitus and its cardiovascular complications,and the biomarkers' value and application of miRNA in the treatment of diabetes mellitus,aiming to develop new insight for the diagnosis and treatment of diabetes mellitus.%miRNA(微小RNA,microRNA)是一类在转录后水平调控基因表达的小分子RNA.近年来多项研究表明miRNA与糖尿病及其多种并发症的发生相关.本文主要就miRNA在糖尿病及其心血管并发症中的调控作用、生物标志物的价值以及在糖尿病治疗中的应用方面作一综述,为糖尿病的诊断及治疗开拓新的思路.

  14. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  15. Powerful regulatory systems and post-transcriptional gene silencing resist increases in cellulose content in cell walls of barley

    OpenAIRE

    Tan, Hwei-Ting; Shirley, Neil J; Singh, Rohan R; Henderson, Marilyn; Dhugga, Kanwarpal S; Mayo, Gwenda M; Fincher, Geoffrey B.; Burton, Rachel A.

    2015-01-01

    Background The ability to increase cellulose content and improve the stem strength of cereals could have beneficial applications in stem lodging and producing crops with higher cellulose content for biofuel feedstocks. Here, such potential is explored in the commercially important crop barley through the manipulation of cellulose synthase genes (CesA). Results Barley plants transformed with primary cell wall (PCW) and secondary cell wall (SCW) barley cellulose synthase (HvCesA) cDNAs driven b...

  16. Study of p53 expression and post-transcriptional modifications after GSM-900 radiofrequency exposure of human amniotic cells.

    Science.gov (United States)

    Bourthoumieu, Sylvie; Magnaudeix, Amandine; Terro, Faraj; Leveque, Philippe; Collin, Alice; Yardin, Catherine

    2013-01-01

    The potential effects of radiofrequency (RF) exposure on the genetic material of cells are very important to determine since genome instability of somatic cells may be linked to cancer development. In response to genetic damage, the p53 protein is activated and can induce cell cycle arrest allowing more time for DNA repair or elimination of damaged cells through apoptosis. The objective of this study was to investigate whether the exposure to RF electromagnetic fields, similar to those emitted by mobile phones of the second generation standard, Global System for Mobile Communications (GSM), may induce expression of the p53 protein and its activation by post-translational modifications in cultured human cells. The potential induction of p53 expression and activation by GSM-900 was investigated after in vitro exposure of human amniotic cells for 24 h to average specific absorption rates (SARs) of 0.25, 1, 2, and 4 W/kg in the temperature range of 36.3-39.7 °C. The exposures were carried out using a wire-patch cell (WPC) under strictly controlled conditions of temperature. Expression and activation of p53 by phosphorylation at serine 15 and 37 were studied using Western blot assay immediately after three independent exposures of cell cultures provided from three different donors. Bleomycin-exposed cells were used as a positive control. According to our results, no significant changes in the expression and activation of the p53 protein by phosphorylation at serine 15 and 37 were found following exposure to GSM-900 for 24 h at average SARs up to 4 W/kg in human embryonic cells.

  17. RNA Expression and Post-Transcriptional Editing Analyses of Cucumber Plastids Reveals Genetic Differences Associated with Chilling Tolerance

    Science.gov (United States)

    Tolerance to chilling injury in cucumber (Cucumis sativus L.) is associated with three plastomic single nucleotide polymorphisms (ptSNPs) at bp positions 4,813, 56,561, and 126,349 that are co-inherited. An understanding of the genetic expression of these ptSNPs as a response to chilling is critical...

  18. 1,25-Dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level

    DEFF Research Database (Denmark)

    Müller, K; Haahr, P M; Diamant, M

    1992-01-01

    (TNF-alpha), produced by the antigen presenting cells. In the present study we examined the effect of 1,25-(OH)2D3 on the production of these cytokines, as well as superoxide generation by freshly isolated mononuclear cells and partially purified monocytes. The immediate precursor of 1,25(OH)2D3, 25-OH...... D3, and the synthetic analogue MC 903 ('Calcipotriol') were examined in parallel. 1,25-(OH)2D3 dose-dependently inhibited the production of IL-alpha, IL-6 and TNF-alpha by Escherichia coli lipopolysaccharide (LPS)-stimulated monocytes, without affecting superoxide production. MC 903 had comparable......1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits lymphocyte proliferation and production of antibodies and lymphokines such as interleukin (IL)-2 and interferon gamma. These lymphocyte functions are dependent upon cytokines, including IL-1 alpha, IL-1 beta, IL-6 and tumour necrosis factor alpha...

  19. Monodehydroascorbate reductase gene, regulated by the wheat PN-2013 miRNA, contributes to adult wheat plant resistance to stripe rust through ROS metabolism.

    Science.gov (United States)

    Feng, Hao; Wang, Xiaojie; Zhang, Qiong; Fu, Yanping; Feng, Chuanxin; Wang, Bing; Huang, Lili; Kang, Zhensheng

    2014-01-01

    Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases worldwide. Varieties with adult plant resistance (APR) maintain effective and durable disease resistance. APR to stripe rust in wheat cultivar XZ9104 (XZ) is associated with extensive hypersensitive cell death and production of reactive oxygen species in the host. MDHAR is an important gene in the AsA-GSH cycle, and it plays an important role in maintaining the reduced pool of AsA scavenging hydrogen peroxide. microRNAs (miRNAs) were shown to engage in post-transcriptional regulation by degrading target mRNAs or repressing gene translation in plants responding to abiotic/biotic stresses. Previously, two novel miRNAs (1136-P3 and PN-2013) were isolated in wheat and the target gene of them was determined using degradome sequencing technology. In this study, the target gene was isolated and characterized as TaMDHAR, a monodehydroascorbate reductase gene. We first demonstrated that the target gene could be cleaved by these two miRNAs in tobacco leaves experimentally. However, TaMDHAR was regulated by PN-2013, not 1136-P3, in wheat-Pst adult incompatible interaction according to the expression patterns. The TaMDHAR knockdown resulted in improved wheat resistance to Pst at the seedling stage, with no influence on 1136-P3 and PN-2013 expression. The TaMDHAR knockdown resulted in a much greater H2O2 accumulation and lower APX and CAT activities together with higher expression in several PR genes. We deduced that TaMDHAR could contribute to the APR of XZ through ROS metabolism as regulated by the AsA-GSH cycle.

  20. Regulation of miR394 in Response to Fusarium oxysporum f. sp. cepae (FOC Infection in Garlic (Allium sativum L

    Directory of Open Access Journals (Sweden)

    Subodh Kumar Chand

    2016-03-01

    Full Text Available MicroRNAs (miRNAs are a class of post transcriptional regulators that negatively regulate gene expression through target mRNA cleavage or translational inhibition and play important roles in plant development and stress response. In the present study, 6 conserved miRNAs from garlic (Allium sativum L. were analysed to identify differentially expressed miRNAs in response to Fusarium oxysporum f. sp. cepae (FOC infection. Stem-loop RT-PCR revealed that miR394 is significantly induced in garlic seedlings post treatment with FOC for 72 h. The induction of miR394 expression during FOC infection was restricted to the basal stem plate tissue, the primary site of infection. Garlic miR394 was also upregulated by exogenous application of jasmonic acid. Two putative targets of miR394 encoding F-box domain and cytochrome P450 (CYP450 family proteins were predicted and verified using 5’ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends assay. Quantitative RT-PCR showed that the transcript levels of the predicted targets were significantly reduced in garlic plants exposed to FOC. When garlic cultivars with variable sensitivity to FOC were exposed to the pathogen, an upregulation of miR394 and down regulation of the targets were observed in both varieties. However, the expression pattern was delayed in the resistant genotypes. These results suggest that miR394 functions in negative modulation of FOC resistance and the difference in timing and levels of expression in variable genotypes could be examined as markers for selection of FOC resistant garlic cultivars.

  1. AB087. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator miR-27b in the spermatozoa of patients with asthenozoospermia

    Science.gov (United States)

    Zhou, Junhao; Zhou, Qizhao; Lyu, Xiaoming; Zhu, Ting; Chen, Zijian; Chen, Mingkun; Xia, Hui; Wang, Chunyan; Qi, Tao; Li, Xin; Liu, Cundong

    2016-01-01

    Objective Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Methods Spermatozoa were isolated from 90 study subjects’ ejaculated semen samples. DNA methylation was evaluated using bisulfite-sequencing PCR and methylation-specific PCR. The CRISP2 mRNA and protein expression levels were examined in the ejaculated spermatozoa by qRT-PCR and Western blot respectively. MiRNA expression was detected by qRT-PCR. The direct regulatory effect of miR-27b on CRISP2 was predicted computationally and validated via luciferase reporter assay and in vitro experiments in which miR-27b mimic or inhibitor was transfected into 293T cells. Respective correlations of miR-27b and CRISP2 protein expression with clinical features were analyzed using Spearman’s correlation coefficient. Results Initially, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic males. Meanwhile, methylation was not found in CRISP2 promoter. These data suggest a possible post-transcriptional regulation of CRISP2 in asthenozoospermia. Subsequently, bioinformatics prediction, luciferase reporter assay and miR-27b transfection experiments revealed that miR-27b could specifically target CRISP2 by binding its 3’-UTR, suppressing CRISP2 expression post-transcriptionally. Further evidence was provided by the clinical observation of a high miR-27b expression in the ejaculated spermatozoa from asthenozoospermic patients and a negative correlation between miR-27b and CRISP2 protein expression. Finally, a retrospective

  2. Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han [Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009 (China); Zhang, Luyong, E-mail: lyzhang@cpu.edu.cn [Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009 (China)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

  3. Regulation of the cell cycle gene, BTG2, by miR-21 in human laryngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Min Liu; Haidong Wu; Tao Liu; Yixuan Li; Fang Wang; Haiying Wan; Xin Li; Hua Tang

    2009-01-01

    MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogenic miRNA, microRNA-21 (miR-21), was found to be upregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the GI-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR-21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oncogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.

  4. MAF2 Is Regulated by Temperature-Dependent Splicing and Represses Flowering at Low Temperatures in Parallel with FLM.

    Directory of Open Access Journals (Sweden)

    Chiara A Airoldi

    Full Text Available Plants enter their reproductive phase when the environmental conditions are favourable for the successful production of progeny. The transition from vegetative to reproductive phase is influenced by several environmental factors including ambient temperature. In the model plant Arabidopsis thaliana, SHORT VEGETATIVE PHASE (SVP is critical for this pathway; svp mutants cannot modify their flowering time in response to ambient temperature. SVP encodes a MADS-box transcription factor that directly represses genes that promote flowering. SVP binds DNA in complexes with other MADS-box transcription factors, including FLOWERING LOCUS M (FLM, which acts with SVP to repress the floral transition at low temperatures. Small temperature changes post-transcriptionally regulate FLM through temperature-dependent alternative splicing (TD-AS. As ambient temperature increases, the predominant FLM splice isoform shifts to encode a protein incapable of exerting a repressive effect on flowering. Here we characterize a closely related MADS-box transcription factor, MADS AFFECTING FLOWERING2 (MAF2, which has independently evolved TD-AS. At low temperatures the most abundant MAF2 splice variant encodes a protein that interacts with SVP to repress flowering. At increased temperature the relative abundance of splice isoforms shifts in favour of an intron-retaining variant that introduces a premature termination codon. We show that this isoform encodes a protein that cannot interact with SVP or repress flowering. At lower temperatures MAF2 and SVP repress flowering in parallel with FLM and SVP, providing an additional input to sense ambient temperature for the control of flowering.

  5. Trout Stream Special Regulations

    Data.gov (United States)

    Minnesota Department of Natural Resources — This layer shows Minnesota trout streams that have a special regulation as described in the 2006 Minnesota Fishing Regulations. Road crossings were determined using...

  6. Hepcidin: regulation of the master iron regulator

    Science.gov (United States)

    Rishi, Gautam; Wallace, Daniel F.; Subramaniam, V. Nathan

    2015-01-01

    Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors. PMID:26182354

  7. General Theories of Regulation

    NARCIS (Netherlands)

    Hertog, J.A. den

    1999-01-01

    This chapter makes a distinction between three types of theories of regulation: public interest theories, the Chicago theory of regulation and the public choice theories. The Chicago theory is mainly directed at the explanation of economic regulation; public interest theories and public choice theor

  8. EST analysis in Ginkgo biloba: an assessment of conserved developmental regulators and gymnosperm specific genes

    Directory of Open Access Journals (Sweden)

    Runko Suzan J

    2005-10-01

    Full Text Available Abstract Background Ginkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate, female (megasporangiate, and vegetative organs (leaves of Ginkgo biloba. Results RNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants – many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads. Conclusion Our analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and

  9. Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues.

    Science.gov (United States)

    Hirota, Takeshi; Date, Yuko; Nishibatake, Yu; Takane, Hiroshi; Fukuoka, Yasushi; Taniguchi, Yuuji; Burioka, Naoto; Shimizu, Eiji; Nakamura, Hiroshige; Otsubo, Kenji; Ieiri, Ichiro

    2012-07-01

    Dihydropyrimidine dehydrogenase (DPD) is important to the antitumor effect of 5-fluorouracil (5-FU). DPD gene (DPYD) expression in tumors is correlated with sensitivity to 5-FU. Because the 5-FU accumulated in cancer cells is also rapidly converted into inactivated metabolites through catabolic pathways mediated by DPD, high DPD activity in cancer cells is an important determinant of the response to 5-FU. DPD activity is highly variable and reduced activity causes a high risk of 5-FU toxicity. Genetic variation in DPYD has been proposed as the main factor responsible for the variation in DPD activity. However, only a small proportion of the activity of DPD can be explained by DPYD mutations. In this study, we found that DPYD is a target of the following microRNAs (miRNA): miR-27a, miR-27b, miR-134, and miR-582-5p. In luciferase assays with HepG2 cells, the overexpression of these miRNAs was associated with significantly decreased reporter activity in a plasmid containing the 3'-UTR of DYPD mRNA. The level of DPD protein in MIAPaca-2 cells was also significantly decreased by the overexpression of these four miRNAs. The results suggest that miR-27a, miR-27b, miR-134, and miR-582-5p post-transcriptionally regulate DPD protein expression. The levels of miRNAs in normal lung tissue and lung tumors were compared; miR-27b and miR-134 levels were significantly lower in the tumors than normal tissue (3.64 ± 4.02 versus 9.75 ± 6.58 and 0.64 ± 0.75 versus 1.48 ± 1.39). DPD protein levels were significantly higher in the tumors. Thus, the decreased expression of miR-27b would be responsible for the high levels of DPD protein. This study is the first to show that miRNAs regulate the DPD protein, and provides new insight into 5-FU-based chemotherapy.

  10. miR-449 inhibits cell proliferation and is down-regulated in gastric cancer

    Directory of Open Access Journals (Sweden)

    Federspiel Birgitte

    2011-03-01

    Full Text Available Abstract Background Gastric cancer is the fourth most common cancer in the world and the second most prevalent cause of cancer related death. The development of gastric cancer is mainly associated with H. Pylori infection leading to a focus in pathology studies on bacterial and environmental factors, and to a lesser extent on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA molecules involved in post-transcriptional gene regulation. They are found to regulate genes involved in diverse biological functions and alterations in microRNA expression have been linked to the pathogenesis of many malignancies. The current study is focused on identifying microRNAs involved in gastric carcinogenesis and to explore their mechanistic relevance by characterizing their targets. Results Invitrogen NCode miRNA microarrays identified miR-449 to be decreased in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared to tissues from wild type animals. Growth rate of gastric cell lines over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell cycle analysis of miR-449 over-expressing cells showed a significant increase in the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We also show that miR-449 over-expression activated p53 and its downstream target p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR analyses showed a loss of miR-449 expression in human clinical gastric tumours compared to normal tissues. Conclusions In this study, we document a diminished expression of miR-449 in Gastrin KO mice and further confirmed its loss in human gastric tumours. We investigated the function of miR-449 by identifying its

  11. Advan ces in Glioma Temozolomide-Resistance Regulated by MicroRNA%MicroRNA调控胶质瘤替莫唑胺化疗耐药的研究进展

    Institute of Scientific and Technical Information of China (English)

    徐勇明; 王宏勤

    2016-01-01

    MicroRNAs (miRNA) are small non-coding RNAs of endogenous small molecules, which are important to regulate the post-transcription of gene expression. MiRNAs in glioma drug resistance has been explored and researched, found that the miRNA plays an important role in drug resistance. Currently, we made a systematic review of the miRNA and Glioma for Temozolomide (TMZ) chemotherapy drug resistance gene for promoting specific pathways in Glioma of new targeted therapy of the further development.%microRNA是一类非编码的内源性小分子RNA,对基因表达进行转录后水平的调控。国内外学者对miRNA在胶质瘤耐药中的作用做了积极的探索和研究,发现miRNA在耐药中起十分重要的作用。本文将阐述miRNA与胶质瘤替莫唑胺化疗耐药基因的关系,促进在胶质瘤中针对特定通路的新靶向治疗的进一步发展。

  12. A Feed-Forward Loop Consisting of the Response Regulator RpaB and the Small RNA PsrR1 Controls Light Acclimation of Photosystem I Gene Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kadowaki, Taro; Nagayama, Ryuta; Georg, Jens; Nishiyama, Yoshitaka; Wilde, Annegret; Hess, Wolfgang R; Hihara, Yukako

    2016-04-01

    Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) have been suggested to be the two most critical factors for this response in Synechocystis sp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of the psrR1 gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence of psrR1 in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and the psrR1 gene is repressed by the binding of RpaB under low-light (LL) conditions. A decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression of psrR1 gene expression. Accumulating PsrR1 molecules then prevent translation from pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions.

  13. Cell-type-specific expression and regulation of a c-fos-NGF fusion gene in neurons and astrocytes of transgenic mice.

    Science.gov (United States)

    Onténiente, B; Horellou, P; Neveu, I; Makeh, I; Suzuki, F; Bourdet, C; Grimber, G; Colin, P; Brachet, P; Mallet, J

    1994-02-01

    A mouse line transgenic for nerve growth factor (NGF) was developed using the mouse prepro-NGF cDNA inserted within a plasmid containing the proximal region (-10 to -550 bp) of the c-fos promoter and the transcription termination and polyadenylation signals of the rabbit beta-globin gene. No significant modification of gross behavior or central nervous system anatomy was detected in adult animals as assessed by immunohistochemistry and in situ hybridization for NGF and choline acetyltransferase. The expression of the transgene and the possible regulation of its expression by agents acting on the promoter were investigated in vitro. Despite the presence of an additional pool of NGF mRNA specific to the transgene, basal levels of NGF in the supernatant of transgenic astrocytes were similar to normal ones. On the other hand, transgenic neurons spontaneously synthesized and released levels of NGF two to three times higher than normal neurons, while mRNA levels were barely detectable by conventional Northern blotting. The tissue-specificity of NGF expression was respected, with higher levels in hippocampal than neocortical neurons. Increases of NGF mRNA by agents acting on the promoter could be observed in normal and transgenic astrocytes only after inhibition of the protein synthesis by cycloheximide, suggesting a similar rapid turnover of normal and transgenic transcripts. Cyclic AMP agonists specifically increased the secretion of NGF protein by transgenic astrocytes and neurons, while activators of the protein kinase C had a similar effect on transgenic and normal cells. Differences between amounts of NGF secreted by neurons and astrocytes with regards to their respective content in mRNA suggest that transgenic transcripts are subject to normal cell- and tissue-specific post-transcriptional regulations. Agents acting on the c-fos promoter through the protein kinase C or cyclic AMP routes differentially increased the secretion of NGF by transgenic astrocytes or

  14. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, plays an important role in cell-to-cell communication during colitis.

    Directory of Open Access Journals (Sweden)

    Saravanan Ayyadurai

    Full Text Available PepT1 is a member of the proton-oligopeptide cotransporter family SLC15, which mediates the transport of di/tripeptides from intestinal lumen into epithelial cells. MicroRNAs (miRNAs, a small noncoding RNAs (21-23 nucleotides, post-transcriptionally regulate gene expression by binding to the 3'-untranslated regions (UTRs of their target mRNAs. Although the role of most miRNAs remains elusive, they have been implicated in vital cellular functions such as intestinal epithelial cells differentiation, proliferation, and apoptosis. In the present study, we investigated the effect of intestinal epithelial PepT1 expression on microRNA (miRNA expression/secretion in the colons of control mice and in mice with experimentally induced colonic inflammation (colitis. The colonic miRNA expression was deregulated in both colitis and control mice but the deregulation of miRNA expression/secretion was specific to colonic tissue and did not affect other tissues such as spleen and liver. Intestinal epithelial PepT1-dependent deregulation of colonic miRNA expression not only affects epithelial cells but also other cell types, such as intestinal macrophages. Importantly, we found the miRNA 23b which was known to be involved in inflammatory bowel disease was secreted and transported between cells to impose a gene-silencing effect on recipient intestinal macrophages. Based on our data, we may conclude that the expression of a specific protein, PepT1, in the intestine affects local miRNA expression/secretion in the colon on a tissue specific manner and may play an important role during the induction and progression of colitis. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, could play a crucial role in cell-to-cell communication during colitis.

  15. The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

    Science.gov (United States)

    Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

    2014-03-01

    CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

  16. Tomato LeTHIC is an Fe-requiring HMP-P synthase involved in thiamine synthesis and regulated by multiple factors.

    Science.gov (United States)

    Zhao, Weina; Cheng, Xudong; Huang, Zongan; Fan, Huajie; Wu, Huilan; Ling, Hong-Qing

    2011-06-01

    Thiamine is a key primary metabolite which is necessary for the viability of all organisms. It is a dietary requirement for mammals because only prokaryotes, fungi and plants are thiamine prototrophs. In contrast to the well documented biosynthetic mechanism in bacteria, much remains to be deciphered in plants. In this work, a tomato thiamine-auxotrophic (thiamineless, tl) mutant was characterized. The tl mutant occurs due to inactivation of LeTHIC transcription as a result of insertion of a large unknown DNA fragment in its 5'-untranslated region. Expression of wild-type LeTHIC in tl plants was able to complement the mutant to wild type. LeTHIC possessed the same function as E.cTHIC [an Escherichia coli 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase involved in synthesis of the pyrimidine moiety of thiamine] because expression of LeTHIC rescued THIC-deficient strains of E. coli under culture conditions without thiamine supplementation, suggesting that plants employ a bacteria-like route of pyrimidine moiety synthesis. LeTHIC is an Fe-S cluster protein localized in chloroplasts, and Fe is required for maintenance of its enzyme activity because Fe deficiency resulted in a significant reduction of thiamine content in tomato leaves. Further, we also showed that the expression of LeTHIC is tightly regulated at the transcriptional and post-transcriptional level by multiple factors, such as light, Fe status and thiamine pyrophosphate (TPP)-riboswitch. The results clearly demonstrated that a feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato. Our results provide a new insight into understanding the molecular mechanism of thiamine biosynthesis in plants.

  17. Small RNA molecules and regulation of spermatogenesis%小RNA分子与精子发生调控

    Institute of Scientific and Technical Information of China (English)

    孟雅楠; 孟丽军; 宋亚娟; 刘美玲; 张秀军

    2011-01-01

    Small RNA molecules (small RNAs) have recently emerged as important regulators of gene expression at the post-transcriptional or translation level.Significant progtess has recently been made in utilizing small RNAs to elucidate the molecular mechanisms regulating spermatogenesis.There are three major small RNAs: small interfering RNAs (siRNAs),microRNAs (miRNAs).and piwi-interacting RNAs (piRNAs).Small-RNAs have diverse biological functions in meiosis and spermatogenesis, In vitro or in vivo, use of siRNA to knockdown genes is a way to study the function of genes of intrest in spermatogenesis.miRNA can be involved in the regulation of mitosis, meiosis, and postmeiosis in spermatogenesis.piRNAs are mainly involved in regulating the process of meiosis and postmeiosis, and repressing retrotransposon transposition in male germline cells.In this paper, we reviewed recent works on the synthesis, mechanism, function, and Outlook of smaIIRNAs.%近来研究发现小RNA(small RNAs)可作为转录后及翻译水平上基因表达调节的重要调节因子,利用小RNA来阐明调节精子发生的分子机制取得了显著进展.这些小RNA主要分为3类,即小干扰RNA(siRNA),微小RNA(miRNA)以及与piwi蛋白相互作用的RNA(piRNA).在减数分裂和精子发生过程中,小RNA具有多种生物学功能,如利用siRNA体外转染或体内注射来敲低特定基因从而研究该基因在精子发生过程中的作用;miRNA可能参与精子发生中有丝、减数及后减数分裂阶段的基因表达调节;piRNA主要参与调节雄性生殖细胞减数及后减数分裂的过程,在精子发生中起抑制反转录转座子(retrotransposons)的作用.文章对小RNAs合成、作用机制、功能及展望等最新进展进行了综述.

  18. The pancreatic and duodenal homeobox protein PDX-1 regulates the ductal specific keratin 19 through the degradation of MEIS1 and DNA binding.

    Directory of Open Access Journals (Sweden)

    Johannes von Burstin

    Full Text Available BACKGROUND: Pancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19. In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH(2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome. CONCLUSIONS/SIGNIFICANCE: Taken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non

  19. TOWARD MORE EFFECTIVE REGULATION

    Energy Technology Data Exchange (ETDEWEB)

    J. GRAF

    2000-06-01

    This paper proposes a model relationship between the operator engaged in a hazardous activity, the regulator of that activity, and the general public. The roles and responsibilities of each entity are described in a way that allows effective communication flow. The role of the regulator is developed using the steam boiler as an example of a hazard subject to regulation; however, the model applies to any regulated activity. In this model the safety analyst has the extremely important role of communicating sometimes difficult technical information to the regulator in a way that the regulator can provide credible assurance to the general public as to the adequacy of the control of the hazardous activity. The conclusion asserts that acceptance of the model, understanding of the roles and responsibilities and definition of who communicates what information to whom will mitigate frustration on the part of each of the three entities.

  20. Novel regulators of spermatogenesis.

    Science.gov (United States)

    Fok, Kin Lam; Chen, Hao; Ruan, Ye Chun; Chan, Hsiao Chang

    2014-05-01

    Spermatogenesis is a multistep process that supports the production of millions of sperm daily. Understanding of the molecular mechanisms that regulate spermatogenesis has been a major focus for decades. Yet, the regulators involved in different cellular processes of spermatogenesis remain largely unknown. Human diseases that result in defective spermatogenesis have provided hints on the molecular mechanisms regulating this process. In this review, we have summarized recent findings on the function and signaling mechanisms of several genes that are known to be associated with disease or pathological processes, including CFTR, CD147, YWK-II and CT genes, and discuss their potential roles in regulating different processes of spermatogenesis.

  1. Epigenetic regulation of L-type voltage-gated Ca(2+) channels in mesenteric arteries of aging hypertensive rats.

    Science.gov (United States)

    Liao, Jingwen; Zhang, Yanyan; Ye, Fang; Zhang, Lin; Chen, Yu; Zeng, Fanxing; Shi, Lijun

    2016-11-24

    Accumulating evidence has shown that epigenetic regulation is involved in hypertension and aging. L-type voltage-gated Ca(2+) channels (LTCCs), the dominant channels in vascular myocytes, greatly contribute to arteriole contraction and blood pressure (BP) control. We investigated the dynamic changes and epigenetic regulation of LTCC in the mesenteric arteries of aging hypertensive rats. LTCC function was evaluated by using microvascular rings and whole-cell patch-clamp in the mesenteric arteries of male Wistar-Kyoto rats and spontaneously hypertensive rats at established hypertension (3 month old) and an aging stage (16 month old), respectively. The expression of the LTCC α1C subunit was determined in the rat mesenteric microcirculation. The expression of miR-328, which targets α1C mRNA, and the DNA methylation status at the promoter region of the α1C gene (CACNA1C) were also determined. In vitro experiments were performed to assess α1C expression after transfection of the miR-328 mimic into cultured vascular smooth muscle cells (VSMCs). The results showed that hypertension superimposed with aging aggravated BP and vascular remodeling. Both LTCC function and expression were significantly increased in hypertensive arteries and downregulated with aging. miR-328 expression was inhibited in hypertension, but increased with aging. There was no significant difference in the mean DNA methylation of CACNA1C among groups, whereas methylation was enhanced in the hypertensive group at specific sites on a CpG island located upstream of the gene promoter. Overexpression of miR-328 inhibited the α1C level of cultured VSMCs within 48 h. The results of the present study indicate that the dysfunction of LTCCs may exert an epigenetic influence at both pre- and post-transcriptional levels during hypertension pathogenesis and aging progression. miR-328 negatively regulated LTCC expression in both aging and hypertension.Hypertension Research advance online publication, 24

  2. Regulation of SUMO Modification

    NARCIS (Netherlands)

    P.M. Knipscheer (Puck Maria)

    2007-01-01

    textabstractThe small ubiquitin related modifier SUMO is a posttranslational modifier that functions in a wide range of cellular processes like intracellular transport, cell cycle regulation, DNA repair and regulation of transcription. SUMO is an 11 kDa protein and is ligated to its target proteins

  3. Reconceptualizing Civil Regulation

    DEFF Research Database (Denmark)

    Galang, Roberto Martin; Castello, Itziar

    2011-01-01

    This article re-conceptualizes the notion of civil regulation, through an analysis of 775 projects by firms located in 21 Asian countries, wherein we map the state of civil regulation initiatives in the region. We challenge two established assumptions in the Corporate Social Responsibility litera...

  4. Plant Growth Regulators.

    Science.gov (United States)

    Nickell, Louis G.

    1978-01-01

    Describes the effect of "plant growth regulators" on plants, such as controlling the flowering, fruit development, plant size, and increasing crop yields. Provides a list of plant growth regulators which includes their chemical, common, and trade names, as well as their different use(s). (GA)

  5. The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

    Directory of Open Access Journals (Sweden)

    Joseph Mazar

    Full Text Available The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3'-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well

  6. MiR-214 regulates the function of osteoblast under simulated microgravity by targeting ATF4

    Science.gov (United States)

    Li, Yingxian; Wang, Xiaogang; Li, Qi; Lv, Ke; Wan, Yumin; Li, Yinghui; Bai, Yanqiang

    Background: MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR) of mRNAs, resulting in translational repression. Growing evidence shows that microRNAs (miRNAs) regu-late various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; How-ever, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast dif-ferentiation remains elusive. Methods: To study the potential involvement of miRNAs in osteoblast differentiation under stimulated microgravity, we analyzed the expression of 20 bone relative miRNAs using real time PCR platform to find particularly miRNAs whose expression is altered during osteoblast differentiation. TargetScan, miRBase and Miranda were used to predict the target gene of candidate miRNA. To investigate whether ATF4 can be directly targeted by miR-214, we engineered luciferase reporters that have either the wild-type 3'UTRs of these genes, or the mutant UTRs with a 6 base pair (bp) deletion in the target sites. Lastly, to address the in vivo role of miR-214 in bone formation, tail suspension mice model was used to simulate the change of osteoblast function and bone loss. Results: Recent studies have sug-gested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify miR-214 in MC3T3-E1 cells, which is a primary mouse osteoblasts cell lin